Methods for detection of genetic disorders

ABSTRACT

The invention provides a method useful for detection of genetic disorders. The method comprises determining the sequence of alleles of a locus of interest, and quantitating a ratio for the alleles at the locus of interest, wherein the ratio indicates the presence or absence of a chromosomal abnormality. The present invention also provides a non-invasive method for the detection of chromosomal abnormalities in a fetus. The invention is especially useful as a non-invasive method for determining the sequence of fetal DNA. The invention further provides methods of isolation of free DNA from a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/661,165, filed Sep. 11, 2003, which is: (a) a continuation-in-part ofPCT/US03/06198, filed Feb. 28, 2003, which claims benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/378,354,filed May 8, 2002, and which is a continuation-in-part of U.S. patentapplication Ser. No. 10/093,618, filed Mar. 11, 2002, which claimsbenefit under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationNo. 60/360,232, filed Mar. 1, 2002; (b) a continuation-in-part ofPCT/US03/27308, filed Aug. 29, 2003; and (c) a continuation-in-part ofU.S. patent application Ser. No. 10/376,770, filed Feb. 28, 2003, whichclaims benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication No. 60/378,354, filed May 8, 2002, and which is acontinuation-in-part of U.S. patent application Ser. No. 10/093,618,filed Mar. 11, 2002, which claims benefit under 35 U.S.C. §119(e) ofU.S. Provisional Patent Application No. 60/360,232, filed Mar. 1, 2002.The contents of these applications are incorporated by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a method for the detection ofgenetic disorders including chromosomal abnormalities and mutations. Thepresent invention provides a rapid, non-invasive method for determiningthe sequence of DNA from a fetus. The method is especially useful fordetection of chromosomal abnormalities in a fetus includingtranslocations, transversions, monosomies, trisomies, and otheraneuploidies, deletions, additions, amplifications, translocations andrearrangements.

2. Background Art

Chromosomal abnormalities are responsible for a significant portion ofgenetic defects in liveborn humans. The nucleus of a human cell containsforty-six (46) chromosomes, which contain the genetic instructions, anddetermine the operations of the cell. Half of the forty-six chromosomesoriginate from each parent. Except for the sex chromosomes, which arequite different from each other in normal males, the chromosomes fromthe mother and the chromosomes from the father make a matched set. Thepairs were combined when the egg was fertilized by the sperm.Occasionally, an error occurs in either the formation or combination ofchromosomes, and the fertilized egg is formed with too many or too fewchromosomes, or with chromosomes that are mixed in some way. Becauseeach chromosome contains many genes, chromosomal abnormalities arelikely to cause serious birth defects, affecting many body systems andoften including developmental disability (e.g., mental retardation).

Cells mistakenly can rejoin broken ends of chromosomes, bothspontaneously and after exposure to chemical compounds, carcinogens, andirradiation. When rejoining occurs within a chromosome, a chromosomesegment between the two breakpoints becomes inverted and is classifiedas an inversion. With inversions, there is no loss of genetic material;however, inversions can cause disruption of a critical gene, or create afusion gene that induces a disease related condition.

In a reciprocal translocation, two non-homologous chromosomes break andexchange fragments. In this scenario, two abnormal chromosomes result:each consists of a part derived from the other chromosome and lacks apart of itself. If the translocation is of a balanced type, theindividual will display no abnormal phenotypes. However, duringgerm-cell formation in the translocation-bearing individuals, the properdistribution of chromosomes in the egg or sperm occasionally fails,resulting in miscarriage, malformation, or mental retardation of theoffspring.

In a Robertsonian translocation, the centromeres of two acrocentric (achromosome with a non-centrally located centromere) chromosomes fuse togenerate one large metacentric chromosome. The karyotype of anindividual with a centric fusion has one less than the normal diploidnumber of chromosomes.

Errors that generate too many or too few chromosomes can also lead todisease phenotypes. For example, a missing copy of chromosome X(monosomy X) results in Turner's Syndrome, while an additional copy ofchromosome 21 results in Down's Syndrome. Other diseases such asEdward's Syndrome, and Patau Syndrome are caused by an additional copyof chromosome 18, and chromosome 13, respectively.

One of the most common chromosome abnormalities is known as Downsyndrome. The estimated incidence of Down's syndrome is between 1 in1,000 to 1 in 1,100 live births. Each year approximately 3,000 to 5,000children are born in the U.S. with this chromosomal disorder. The vastmajority of children with Down syndrome (approximately 95 percent) havean extra chromosome 21. Most often, the extra chromosome originates fromthe mother. However, in about 3-4 percent of people with Down syndrome,a translocation between chromosome 21 and either 14 or 22 is responsiblefor the genetic abnormality. Finally, another chromosome problem, calledmosaicism, is noted in about 1 percent of individuals with Down'ssyndrome. In this case, some cells have 47 chromosomes and others have46 chromosomes. Mosaicism is thought to be the result of an error incell division soon after conception.

Chromosomal abnormalities are congenital, and therefore, prenataldiagnosis can be used to determine the health and condition of an unbornfetus. Without knowledge gained by prenatal diagnosis, there could be anuntoward outcome for the fetus or the mother or both. Congenitalanomalies account for 20 to 25% of perinatal deaths. Specifically,prenatal diagnosis is helpful for managing the remaining term of thepregnancy, planning for possible complications with the birth process,preparing for problems that can occur in the newborn infant, and findingconditions that may affect future pregnancies.

There are a variety of non-invasive and invasive techniques availablefor prenatal diagnosis including ultrasonography, amniocentesis,chorionic villus sampling (CVS), fetal blood cells in maternal blood,maternal serum alpha-fetoprotein, maternal serum beta-HCG, and maternalserum estriol. However, the techniques that are non-invasive are lessspecific, and the techniques with high specificity and high sensitivityare highly invasive. Furthermore, most techniques can be applied onlyduring specific time periods during pregnancy for greatest utility.

Ultrasonography

This is a harmless, non-invasive procedure. High frequency sound wavesare used to generate visible images from the pattern of the echoes madeby different tissues and organs, including the fetus in the amnioticcavity. The developing embryo can be visualized at about 6 weeks ofgestation. The major internal organs and extremities can be assessed todetermine if any are abnormal at about 16 to 20 weeks gestation.

An ultrasound examination can be useful to determine the size andposition of the fetus, the amount of amniotic fluid, and the appearanceof fetal anatomy; however, there are limitations to this procedure.Subtle abnormalities, such as Down syndrome, where the morphologicabnormalities are often not marked, but only subtle, may not be detectedat all.

Amniocentesis

This is a highly invasive procedure in which a needle is passed throughthe mother's lower abdomen into the amniotic cavity inside the uterus.This procedure can be performed at about 14 weeks gestation. Forprenatal diagnosis, most amniocenteses are performed between 14 and 20weeks gestation. However, an ultrasound examination is performed, priorto amniocentesis, to determine gestational age, position of the fetusand placenta, and determine if enough amniotic fluid is present. Withinthe amniotic fluid are fetal cells (mostly derived from fetal skin)which can be grown in culture for chromosomal, biochemical, andmolecular biologic analyses.

Large chromosomal abnormalities, such as extra or missing chromosomes orchromosome fragments, can be detected by karyotyping, which involves theidentification and analysis of all 46 chromosomes from a cell andarranges them in their matched pairs, based on subtle differences insize and structure. In this systematic display, abnormalities inchromosome number and structure are apparent. This procedure typicallytakes 7-10 days for completion.

While amniocentesis can be used to provide direct genetic information,risks are associated with the procedure including fetal loss andmaternal Rh sensitization. The increased risk for fetal mortalityfollowing amniocentesis is about 0.5% above what would normally beexpected. Rh negative mothers can be treated with RhoGam.

Chorionic Villus Sampling (CVS)

In this procedure, a catheter is passed via the vagina through thecervix and into the uterus to the developing placenta with ultrasoundguidance. The introduction of the catheter allows cells from theplacental chorionic villi to be obtained and analyzed by a variety oftechniques, including chromosome analysis to determine the karyotype ofthe fetus. The cells can also be cultured for biochemical or molecularbiologic analysis. Typically, CVS is performed between 9.5 and 12.5weeks gestation.

CVS has the disadvantage of being an invasive procedure, and it has alow but significant rate of morbidity for the fetus; this loss rate isabout 0.5 to 1% higher than for women undergoing amniocentesis. Rarely,CVS can be associated with limb defects in the fetus. Also, thepossibility of maternal Rh sensitization is present. Furthermore, thereis also the possibility that maternal blood cells in the developingplacenta will be sampled instead of fetal cells and confound chromosomeanalysis.

Maternal Serum Alpha-Fetoprotein (MSAFP)

The developing fetus has two major blood proteins—albumin andalpha-fetoprotein (AFP). The mother typically has only albumin in herblood, and thus, the MSAFP test can be utilized to determine the levelsof AFP from the fetus. Ordinarily, only a small amount of AFP gainsaccess to the amniotic fluid and crosses the placenta to mother's blood.However, if the fetus has a neural tube defect, then more AFP escapesinto the amniotic fluid. Neural tube defects include anencephaly(failure of closure at the cranial end of the neural tube) and spinabifida (failure of closure at the caudal end of the neural tube). Theincidence of such defects is about 1 to 2 births per 1000 in the UnitedStates. Also, if there are defects in the abdominal wall, the AFP fromthe fetus will end up in maternal blood in higher amounts.

The amount of MSAFP increases with gestational age, and thus for theMSAFP test to provide accurate results, the gestational age must beknown with certainty. Also, the race of the mother and presence ofgestational diabetes can influence the level of MSAFP that is to beconsidered normal. The MSAFP is typically reported as multiples of themean (MoM). The greater the MoM, the more likely a defect is present.The MSAFP test has the greatest sensitivity between 16 and 18 weeksgestation, but can be used between 15 and 22 weeks gestation. The MSAFPtends to be lower when Down's Syndrome or other chromosomalabnormalities is present.

While the MSAFP test is non-invasive, the MSAFP is not 100% specific.MSAFP can be elevated for a variety of reasons that are not related tofetal neural tube or abdominal wall defects. The most common cause foran elevated MSAFP is a wrong estimation of the gestational age of thefetus. Therefore, results from an MSAFP test are never considereddefinitive and conclusive.

Maternal Serum Beta-HCG

Beginning at about a week following conception and implantation of thedeveloping embryo into the uterus, the trophoblast will producedetectable beta-HCG (the beta subunit of human chorionic gonadotropin),which can be used to diagnose pregnancy. The beta-HCG also can bequantified in maternal serum, and this can be useful early in pregnancywhen threatened abortion or ectopic pregnancy is suspected, because theamount of beta-HCG will be lower than normal.

In the middle to late second trimester, the beta-HCG can be used inconjunction with the MSAFP to screen for chromosomal abnormalities, inparticular for Down syndrome. An elevated beta-HCG coupled with adecreased MSAFP suggests Down syndrome. High levels of HCG suggesttrophoblastic disease (molar pregnancy). The absence of a fetus onultrasonography along with an elevated HCG suggests a hydatidiform mole.

Maternal Serum Estriol

The amount of estriol in maternal serum is dependent upon a viablefetus, a properly functioning placenta, and maternal well-being.Dehydroepiandrosterone (DHEA) is made by the fetal adrenal glands, andis metabolized in the placenta to estriol. The estriol enters thematernal circulation and is excreted by the maternal kidney in urine orby the maternal liver in the bile. Normal levels of estriol, measured inthe third trimester, will give an indication of general well-being ofthe fetus. If the estriol level drops, then the fetus is threatened andan immediate delivery may be necessary. Estriol tends to be lower whenDown syndrome is present and when there is adrenal hypoplasia withanencephaly.

The Triple Screen Test

The triple screen test comprises analysis of maternal serumalpha-feto-protein (MSAFP), human chorionic gonadotrophin (hCG), andunconjugated estriol (uE3). The blood test is usually performed 16-18weeks after the last menstrual period. While the triple screen test isnon-invasive, abnormal test results are not indicative of a birthdefect. Rather, the test only indicates an increased risk and suggeststhat further testing is needed. For example, 100 out of 1,000 women willhave an abnormal result from the triple screen test. However, only 2-3of the 100 women will have a fetus with a birth defect. This highincidence of false positives causes tremendous stress and unnecessaryanxiety to the expectant mother.

Fetal Cells Isolated from Maternal Blood

The presence of fetal nucleated cells in maternal blood makes itpossible to use these cells or noninvasive prenatal diagnosis(Walknowska, et al., Lancet 1:1119-1122, 1969; Lo et al., Lancet2:1363-65, 1989; Lo et al., Blood 88:4390-95, 1996). The fetal cells canbe sorted and analyzed by a variety of techniques to look for particularDNA sequences (Bianchi et al., Am. J. Hum. Genet. 61:822-29, (1997);Bianchi et al., PNAS 93:705-08, (1996)). Fluorescence in-situhybridization (FISH) is one technique that can be applied to identifyparticular chromosomes of the fetal cells recovered from maternal bloodand diagnose aneuploid conditions such as trisomies and monosomy X.Also, it has been reported that the number of fetal cells in maternalblood increases in aneuploid pregnancies.

The method of FISH uses DNA probes labeled with colored fluorescent tagsthat allow detection of specific chromosomes or genes under amicroscope. Using FISH, subtle genetic abnormalities that cannot bedetected by standard karyotyping are readily identifiable. Thisprocedure typically takes 24-48 hours to complete. Additionally, using apanel of multi-colored DNA FISH probes, abnormal chromosome copy numberscan be seen.

While improvements have been made for the isolation and enrichment offetal cells, it is still difficult to get many fetal blood cells. Theremay not be enough to reliably determine anomalies of the fetal karyotypeor assay for other abnormalities. Furthermore, most techniques are timeconsuming, require high-inputs of labor, and are difficult to implementfor a high throughput fashion.

Fetal DNA from Maternal Blood

Fetal DNA has been detected and quantitated in maternal plasma and serum(Lo et al., Lancet 350:485-487 (1997); Lo et al., Am. J. hum. Genet.62:768-775 (1998)). Multiple fetal cell types occur in the maternalcirculation, including fetal granulocytes, lymphocytes, nucleated redblood cells, and trophoblast cells (Pertl, and Bianchi, Obstetrics andGynecology 98: 483-490 (2001)). Fetal DNA can be detected in the serumat the seventh week of gestation, and increases with the term of thepregnancy. The fetal DNA present in the maternal serum and plasma iscomparable to the concentration of DNA obtained from fetal cellisolation protocols.

Circulating fetal DNA has been used to determine the sex of the fetus(Lo et al., Am. J. hum. Genet. 62:768-775 (1998)). Also, fetal rhesus Dgenotype has been detected using fetal DNA. However, the diagnostic andclinical applications of circulating fetal DNA is limited to genes thatare present in the fetus but not in the mother (Pertl and Bianchi,Obstetrics and Gynecology 98: 483-490 (2001)). Thus, a need still existsfor a non-invasive method that can determine the sequence of fetal DNAand provide definitive diagnosis of chromosomal abnormalities in afetus.

BRIEF SUMMARY OF THE INVENTION

The invention is directed to a method for detection of genetic disordersincluding mutations and chromosomal abnormalities. In some embodiments,the present invention is used to detect mutations, and chromosomalabnormalities including but not limited to translocation, transversion,monosomy, trisomy, and other aneuploidies, deletion, addition,amplification, fragment, translocation, and rearrangement. Numerousabnormalities can be detected simultaneously. The present invention alsoprovides a non-invasive method to determine the sequence of fetal DNAfrom a sample of a pregnant female. The present invention can be used todetect any alternation in gene sequence as compared to the wild typesequence including but not limited to point mutation, reading frameshift, transition, transversion, addition, insertion, deletion,addition-deletion, frame-shift, missense, reverse mutation, andmicrosatellite alteration. The present invention also provides a methodfor isolating free nucleic acid from a sample containing nucleic acid.The present invention also provides compositions and kits.

In one aspect, the invention is directed to methods for detectingchromosomal abnormalities. In one embodiment, the present invention isdirected to a method for detecting chromosomal abnormalities, saidmethod comprising quantitating the relative amount of the alleles at aheterozygous locus of interest, where the heterozygous locus of interestwas previously identified by determining the sequence of alleles at alocus of interest from template DNA, wherein said relative amount isexpressed as a ratio, and wherein said ratio indicates the presence orabsence of a chromosomal abnormality.

In some embodiments, determining the sequence includes using a methodthat is allele specific PCR, mass spectrometry, hybridization, primerextension, fluorescence resonance energy transfer (FRET), sequencing,Sanger dideoxy sequencing, DNA microarray, GeneCHIP arrays, HuSNParrays, CodeLink Arrays, BeadArray Technology, MassARRAY, MassEXTEND,SNP-IT, TaqMan, InvaderStrand Assay, southern blot, slot blot, dot blot,or MALDI-TOF mass spectrometry.

In some embodiments, template DNA is obtained from human, non-human,mammal, reptile, cattle, cat, dog, goat, swine, pig, monkey, ape,gorilla, bull, cow, bear, horse, sheep, poultry, mouse, rat, fish,dolphin, whale, or shark. In an embodiment, the template DNA is obtainedfrom a human source. In a preferred embodiment, the template DNA isobtained from a pregnant human female. In some embodiments, the templateDNA is obtained from a sample that is a cell, fetal cell, tissue, blood,serum, plasma, saliva, urine, tear, vaginal secretion, sweat, umbilicalcord blood, chorionic villi amniotic fluid, embryonic tissue, an embryo,a two-celled embryo, a four-celled embryo, an eight celled embryo, a16-celled embryo, a 32-celled embryo, a 64-celled embryo, a 128-celledembryo, a 256-celled embryo, a 512-celled embryo, a 1024-celled embryo,lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid,ascitic fluid, fecal matter, or body exudates. In these embodiments, thesample may be mixed with an agent that inhibits cell lysis to inhibitthe lysis of cells, if cells are present, where the agent is a membranestabilizer, a cross-linker, or a cell lysis inhibitor. In some of theseembodiments, agent is a cell lysis inhibitor, and may be glutaraldehyde,derivatives of glutaraldehyde, formaldehyde, formalin, or derivatives offormaldehyde. In some of these embodiments the sample is blood and inone embodiment the sample is blood from a pregnant female, e.g., a humanfemale. In the latter embodiment, the fetus may be at a gestational ageselected from the group consisting of: 0-4, 4-8, 8-12, 12-16, 16-20,20-24, 24-28, 28-32, 32-36, 36-40, 40-44, 44-48, 48-52, or more than 52weeks. In some of these embodiments, the template DNA may be obtainedfrom plasma or from serum from the blood. In these embodiments, thetemplate DNA may include a mixture of maternal DNA and fetal DNA, and inone embodiment, prior to determining the sequence of alleles of a locusof interest from template DNA, maternal DNA is sequenced to identify ahomozygous locus of interest, and the homozygous locus of interest isthe locus of interest analyzed in the template DNA. In anotherembodiment, maternal DNA is sequenced to identify a heterozygous locusof interest, and the heterozygous locus of interest is the locus ofinterest analyzed in the template DNA.

In embodiments, alleles of multiple loci of interest are sequenced andtheir relative amounts quantitated and expressed as a ratio. In oneembodiment, the sequence of alleles of one to tens to hundreds tothousands of loci of interest on a single chromosome on template DNA isdetermined. In another embodiment, the sequence of alleles of one totens to hundreds to thousands of loci of interest on multiplechromosomes is determined.

In an embodiment, the locus of interest is suspected of containing asingle nucleotide polymorphism or mutation. The method can be used fordetermining sequences of multiple loci of interest concurrently. Thetemplate DNA can comprise multiple loci from a single chromosome. Thetemplate DNA can comprise multiple loci from different chromosomes. Theloci of interest on template DNA can be amplified in one reaction.Alternatively, each of the loci of interest on template DNA can beamplified in a separate reaction. The amplified DNA can be pooledtogether prior to digestion of the amplified DNA. Each of the labeledDNA containing a locus of interest can be separated prior to determiningthe sequence of the locus of interest. In one embodiment, at least oneof the loci of interest is suspected of containing a single nucleotidepolymorphism or a mutation.

There is no limitation as to the chromosomes that can be compared. Theratio for the alleles at a heterozygous locus of interest on anychromosome can be compared to the ratio for the alleles at aheterozygous locus of interest on any other chromosome. In anotherembodiment, the ratio of alleles at a heterozygous locus of interest ona chromosome is compared to the ratio of alleles at a heterozygous locusof interest on two, three, four or more than four chromosomes. Inanother embodiment, the ratio of alleles at multiple loci of interest ona chromosome is compared to the ratio of alleles at multiple loci ofinterest on two, three, four, or more than four chromosomes. Inembodiments, the ratio for alleles at heterozygous loci of interest on achromosome are summed and compared to the ratio for alleles atheterozygous loci of interest on a different chromosome, where adifference in ratios indicates the presence of a chromosomalabnormality. In some of these embodiments, the chromosomes that arecompared are human chromosomes such as chromosome 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, or Y. Inone of the latter embodiments, the ratio for the alleles at heterozygousloci of interest of chromosomes 13, 18, and 21 are compared. In anotherembodiment, the sequence of one to tens to hundreds to thousands of lociof interest on the template DNA obtained from a sample of a pregnantfemale is determined. In one embodiment, the loci of interest are on onechromosome. In another embodiment, the loci of interest are on multiplechromosomes.

In some embodiments, determining the sequence of the alleles comprisesamplifying alleles of a locus of interest on a template DNA using afirst and a second primer, where the second primer contains arecognition site for a restriction enzyme such that digestion with therestriction enzyme generates a 5′ overhang containing the locus ofinterest; digesting the amplified DNA with the restriction enzyme thatrecognizes the recognition site on the second primer; incorporating anucleotide into the digested DNA by using the 5′ overhang containing thelocus of interest as a template; and determining the sequence of thealleles of the locus of interest by determining the sequence of the DNAinto which the nucleotide was incorporated. In one embodiment,determination of the sequence of the locus of interest in comprisesdetecting a nucleotide.

In other embodiments, determining the sequence of alleles comprisesamplifying alleles of a locus of interest on a template DNA using afirst and second primers, where the second primer contains a recognitionsite for a restriction enzyme such that digestion with the restrictionenzyme generates a 5′ overhang containing the locus of interest;digesting the amplified DNA with the restriction enzyme that recognizesthe recognition site on the second primer; incorporating nucleotidesinto the digested DNA of (b), where a nucleotide that terminateselongation, and is complementary to the locus of interest of an allele,is incorporated into the 5′ overhang of said allele, and a nucleotidecomplementary to the locus of interest of a different allele isincorporated into the 5′ overhang of said different allele, and saidterminating nucleotide, which is complementary to a nucleotide in the 5′overhang of said different allele, is incorporated into the 5′ overhangof said different allele; and determining the sequence of the alleles ofa locus of interest by determining the sequence of the DNA into whichthe complementary nucleotides have been incorporated. In one embodiment,determination of the sequence of the locus of interest comprisesdetecting a nucleotide.

The incorporation of a nucleotide may be accomplished by a DNApolymerase, including but not limited to E. coli DNA polymerase, Klenowfragment of E. coli DNA polymerase I, T7 DNA polymerase, T4 DNApolymerase, T5 DNA polymerase, Klenow class polymerases, Taq polymerase,bacteriophage 29, REDTaq™ Genomic DNA polymerase, Pfu DNA polymerase,Vent DNA polymerase or sequenase. Incorporation of a nucleotide mayinclude incorporation of a labeled nucleotide, or labeled and unlabelednucleotides. One nucleotide, two nucleotides, three nucleotides, fournucleotides, five nucleotides, or more than five nucleotides can beincorporated. A combination of labeled and unlabeled nucleotides can beincorporated. The labeled nucleotide may be a dideoxynucleotidetriphosphate (also referred to as “dideoxy”) or deoxynucleotidetriphosphate (also referred to as “deoxy”). The unlabeled nucleotide maybe a dideoxynucleotide triphosphate or deoxynucleotide triphosphate.Labeled nucleotides may be labeled with a radioactive molecule,fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate,biotin, derivative of biotin, phosphorescent moiety, luminescent moiety,electrochemiluminescent moiety, chromatic moiety, and moiety having adetectable electron spin resonance, electrical capacitance, dielectricconstant or electrical conductivity. In one embodiment, the labelednucleotide is labeled with a fluorescent molecule. The incorporation ofa fluorescent labeled nucleotide may further comprise using a mixture offluorescent and unlabeled nucleotides.

In one embodiment, the determination of the sequence of the locus ofinterest comprises detecting the incorporated nucleotide. The detectionmethod includes but is not limited to gel electrophoresis, capillaryelectrophoresis, microchannel electrophoresis, polyacrylamide gelelectrophoresis, fluorescence detection, fluorescence polarization, DNAsequencing, Sanger dideoxy sequencing, ELISA, mass spectrometry, time offlight mass spectrometry, quadrupole mass spectrometry, magnetic sectormass spectrometry, electric sector mass spectrometry, fluorometry,infrared spectrometry, ultraviolet spectrometry, palentiostaticamperometry, DNA hybridization, DNA microarray, GeneChip arrays, HuSNParrays, BeadArrays, MassExtend, SNP-IT, TaqMan assay, Invader assay,MassCleave, southern blot, slot blot, or dot blot.

In embodiments, first and second primers contain a portion of arestriction enzyme recognition site that contains a variable nucleotide,where the full restriction enzyme recognition site is generated afteramplification. In some embodiments, the 3′ region of said primers cancontain mismatches with the template DNA, and digestion with saidrestriction enzyme generates a 5′ overhang containing the locus ofinterest. In some embodiments, the restriction enzyme recognition siteis for a restriction enzyme that includes but is not limited to BsaJ I,Bssk I, Dde I, EcoN I, Fnu4H I, Hinf I, or ScrF I. In some embodiments,the restriction enzyme cuts DNA at a distance, from the recognitionsite. In some of these embodiments, the recognition site is for a TypeIIS restriction enzyme. In some of these embodiments, the Type IISrestriction enzyme includes but is not limited to Alw I, Alw26 I, Bbs I,Bbv I, BceA I, Bmr I, Bsa I, Bst71 I, BsmA I, BsmB I, BsmF I, BspM I,Ear I, Fau I, Fok I, Hga I, Ple I, Sap I, SSfaN I, or Sthi32 I.

In some embodiments, the recognition site for restriction enzymesincludes but is not limited to BsaJ I (5′ C^(↓)CNNGG 3′), BssK I(5′^(↓)CCNGG 3′), Dde I (5′C^(↓)TNAG 3′), EcoN I (5′CCTNN^(↓)NNNAGG 3′(SEQ ID NO: 7)), Fnu4H I (5′GC^(↓)NGC 3′), Hinf I (5′G^(↓)ANTC 3′), PflFI (5′ GACN^(↓)NNGTC 3′), Sau96 I (5′ G^(↓)GNCC 3′), ScrF I (5′ CC^(↓)NGG3′), Tth1 11 I (5′ GACN^(↓)NNGTC 3′), and more preferably Fnu4H I andEcoN I, is generated after amplification.

The first and/or second primer can contain a tag at the 5′ terminus. Insome embodiments, the first primer contains a tag at the 5′ terminus.The tag can be used to separate the amplified DNA from the template DNA.The tag can be used to separate the amplified DNA containing the labelednucleotide from the amplified DNA that does not contain the labelednucleotide. The tag can be any chemical moiety including but not limitedto radioisotope, fluorescent reporter molecule, chemiluminescentreporter molecule, antibody, antibody fragment, hapten, biotin,derivative of biotin, photobiotin, iminobiotin, digoxigenin, avidin,enzyme, acridinium, sugar, enzyme, apoenzyme, homopolymericoligonucleotide, hormone, ferromagnetic moiety, paramagnetic moiety,diamagnetic moiety, phosphorescent moiety, luminescent moiety,electrochemiluminescent moiety, chromatic moiety, moiety having adetectable electron spin resonance, electrical capacitance, dielectricconstant or electrical conductivity, or combinations thereof. In someembodiments, the tag is biotin. The biotin tag is used to separateamplified DNA from the template DNA using a streptavidin matrix. Thestreptavidin matrix may be coated on wells of a microtiter plate.

In some embodiments, the annealing length of the second primer isselected from the group consisting of 35-30, 30-25, 25-20, 20-15, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, and less than 4 bases.

In embodiments, the method of amplification includes but is not limitedto polymerase chain reaction, self-sustained sequence reaction, ligasechain reaction, rapid amplification of cDNA ends, polymerase chainreaction and ligase chain reaction, Q-beta phage amplification, stranddisplacement amplification, or splice overlap extension polymerase chainreaction. In some embodiments, the method of amplification is PCR. Insome of these embodiments, an annealing temperature for cycle 1 of PCRis about the melting temperature of the portion of the 3′ region of thesecond primer that anneals to the template DNA. In some of the latterembodiments, an annealing temperature for cycle 2 of PCR is about themelting temperature of the portion of the 3′ region of the first primerthat anneals to the template DNA. In some of the latter embodiments, anannealing temperature for the remaining cycles of PCR is at about themelting temperature of the entire second primer.

In another aspect, the invention provides methods of determining thesequence of a locus of interest from a sample comprising free fetal DNA,where an agent that inhibits cell lysis has been added to the sample toinhibit lysis of cells, if cells are present, where the agent is amembrane stabilizer, a cross-linker, or a cell lysis inhibitor.

In some embodiments, the agent is a cell lysis inhibitor, and in some ofthese embodiments, the cell lysis inhibitor includes but is not limitedto glutaraldehyde, derivatives of glutaraldehyde, formaldehyde,derivatives of formaldehyde, or formalin. In embodiments, the sampleincludes but is not limited to tissue, cell, blood, serum, plasma,urine, or vaginal secretion. In some embodiments, the sample is blood.In some of these embodiments, the template DNA is isolated from theserum, in other embodiments the template DNA is isolated from plasma. Insome embodiments, the sample contains free maternal template DNA andfree fetal template DNA. In some embodiments, prior to determining thesequence, template DNA was isolated. In some embodiments, prior todetermining the sequence of the locus of interest on fetal DNA, thesequence of the locus of interest on maternal template DNA wasdetermined. In some embodiments, prior to determining the sequence ofthe locus of interest on fetal DNA, the sequence of the locus ofinterest on paternal template DNA was determined. In some embodiments,the locus of interest is a single nucleotide polymorphism. In otherembodiments, the locus of interest is a mutation. In some embodiments,the sequence of multiple loci of interest is determined. In some ofthese embodiments, the multiple loci of interest are on multiplechromosomes.

In some embodiments, the sequence is determined by: (a) amplifying alocus of interest on a template DNA using a first and second primers,where the second primer contains a recognition site for a restrictionenzyme such that digestion with the restriction enzyme generates a 5′overhang containing the locus of interest; (b) digesting the amplifiedDNA with the restriction enzyme that recognizes the recognition site onthe second primer; (c) incorporating a nucleotide into the digested DNAof (b) by using the 5′ overhang containing the locus of interest as atemplate; and (d) determining the sequence of the locus of interest bydetermining the sequence of the DNA of (c).

In other embodiments, the sequence is determined by: (a) amplifyingalleles of a locus of interest on a template DNA using a first andsecond primers, where the second primer contains a recognition site fora restriction enzyme such that digestion with the restriction enzymegenerates a 5′ overhang containing the locus of interest; (b) digestingthe amplified DNA with the restriction enzyme that recognizes therecognition site on the second primer; (c) incorporating nucleotidesinto the digested DNA of (b), where a nucleotide that terminateselongation, and is complementary to the locus of interest of an allele,is incorporated into the 5′ overhang of said allele, and a nucleotidecomplementary to the locus of interest of a different allele isincorporated into the 5′ overhang of said different allele, and theterminating nucleotide, which is complementary to a nucleotide in the 5′overhang of said different allele, is incorporated into the 5′ overhangof said different allele; and (d) determining the sequence of thealleles of a locus of interest by determining the sequence of the DNA of(c).

In some embodiments, the restriction enzyme cuts DNA at a distance fromthe recognition site. In some of these embodiments, the recognition siteincludes but is for a Type IIS restriction enzyme, for example Alw I,Alw26 I, Bbs I, Bbv I, BceA I, Bmr I, Bsa I, Bst71 I, BsmA I, BsmB I,BsmF I, BspM I, Ear I, Fau I, Fok I, Hga I, Ple I, Sap I, SSfaN I, orSthi32 I.

In some embodiments, the method of amplification may be, for example,polymerase chain reaction, self-sustained sequence reaction, ligasechain reaction, rapid amplification of cDNA ends, polymerase chainreaction and ligase chain reaction, Q-beta phage amplification, stranddisplacement amplification, or splice overlap extension polymerase chainreaction. In some embodiments, the method of amplification is by PCR. Insome of these embodiments, an annealing temperature for cycle 1 of PCRis about the melting temperature of the portion of the 3′ region of thesecond primer that anneals to the template DNA. In some of the latterembodiments, an annealing temperature for cycle 2 of PCR is about themelting temperature of the portion of the 3′ region of the first primerthat anneals to the template DNA. In some of the latter embodiments, anannealing temperature for the remaining cycles of PCR is at about themelting temperature of the entire second primer.

In some embodiments, the sequence of a locus of interest was determinedusing allele specific PCR, mass spectrometry, hybridization, primerextension, fluorescence polarization, fluorescence resonance energytransfer (FRET), fluorescence detection, sequencing, Sanger dideoxysequencing, DNA microarray, southern blot, slot blot, dot blot, orMALDI-TOF mass spectrometry.

In some embodiments, the sequence of a locus of interest is determinedby (1) amplification of the locus of interest; (2) hybridization ofamplified loci to GeneCHIP array (3) washing GeneCHIP array; (4)staining the GeneCHIP array with detectable reagents; and (5) scanningGeneCHIP array. In some of these embodiments, the amplification methodin (1) is polymerase chain reaction, self-sustained sequence reaction,ligase chain reaction, rapid amplification of cDNA ends, polymerasechain reaction and ligase chain reaction, Q-beta phage amplification,strand displacement amplification, or splice overlap extensionpolymerase chain reaction. In some embodiments, the method ofamplification is by PCR. In some embodiments, the staining methodcomprises streptavidin phycoerythrin and biotinylated anti-streptavidin.In some embodiments, an agent that inhibits cell lysis has been added tothe sample to inhibit the lysis of cells, if present, where the agent ismembrane stabilizer, cross-linker, or cell lysis inhibitor. In someembodiments, the agent is a cell lysis inhibitor. In some of theseembodiments, the cell lysis inhibitor is formalin at a percentageselected from the group consisting of: 0.0001-0.03%, 0.03-0.05%,0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%,1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration offormalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determinedby (1) amplification of the locus of interest; (2) ampliconfragmentation; (3) hybridization of fragmented amplicons to CodeLinkArrays; (4) extension reaction to incorporate a nucleotide; and (5)detection of incorporated nucleotides. In some of these embodiments, theamplification method is polymerase chain reaction, self-sustainedsequence reaction, ligase chain reaction, rapid amplification of cDNAends, polymerase chain reaction and ligase chain reaction, Q-beta phageamplification, strand displacement amplification, or splice overlapextension polymerase chain reaction. In some embodiments, the method ofamplification is by PCR. In some embodiments, the amplicon fragmentationis by exonuclease digestion. In some embodiments, the incorporatednucleotide is a dideoxynucleotide or deoxynucleotide. In someembodiments, the incorporated nucleotide is labeled with a radioactivemolecule, fluorescent molecule, antibody, antibody fragment, hapten,carbohydrate, biotin, derivative of biotin, phosphorescent moiety,luminescent moiety, electrochemiluminescent moiety, chromatic moiety,and moiety having a detectable electron spin resonance, electricalcapacitance, dielectric constant or electrical conductivity. In someembodiments, the labeled nucleotide is labeled with a fluorescentmolecule. In some embodiments, an agent that inhibits cell lysis hasbeen added to the sample to inhibit the lysis of cells, if present,where the agent is membrane stabilizer, cross-linker, or cell lysisinhibitor. In some embodiments, the agent is a cell lysis inhibitor. Insome of these embodiments, the cell lysis inhibitor is formalin at apercentage selected from the group consisting of: 0.0001-0.03%,0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%,0.7-0.9%, 0.9-1.2%, 1.5-2%, or 2-3%. In some embodiments, theconcentration of formalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determinedby using BeadArray Technology. In some embodiments, an agent thatinhibits cell lysis has been added to the sample to inhibit the lysis ofcells, if present, where the agent is membrane stabilizer, cross-linker,or cell lysis inhibitor. In some embodiments, the agent is a cell lysisinhibitor. In some of these embodiments, the cell lysis inhibitor isformalin at a percentage selected from the group consisting of:0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%,0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In someembodiments, the concentration of formalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determinedby (1) amplification of the locus of interest; (2) dephosphorylation ofthe unused reagents in (1); (3) in vitro transcription reaction of theproducts of (2); (4) RNase A cleavage of the products of (3); (5) mixingthe products of (4) with CleanResin; (6) transfer products of (5) toSpectroCHIP; and (7) analysis of the SpectroCHIP. In some of theseembodiments, the amplification method is polymerase chain reaction,self-sustained sequence reaction, ligase chain reaction, rapidamplification of cDNA ends, polymerase chain reaction and ligase chainreaction, Q-beta phage amplification, strand displacement amplification,or splice overlap extension polymerase chain reaction. In someembodiments, the method of amplification is by PCR. In some embodiments,the dephosphorylation reaction is catalyzed by shrimp alkalinephosphatase. In some embodiments, an agent that inhibits cell lysis hasbeen added to the sample to inhibit the lysis of cells, if present,where the agent is membrane stabilizer, cross-linker, or cell lysisinhibitor. In some embodiments, the agent is a cell lysis inhibitor. Insome of these embodiments, the cell lysis inhibitor is formalin at apercentage selected from the group consisting of 0.0001-0.03%,0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%,0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, theconcentration of formalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determinedby (1) amplification of a locus of interest; (2) dephosphorylation ofthe unused reagents in (1); (3) hybridization of a primer to the locusof interest; (4) incorporation of a nucleotide; (5) mixing the productsof (4) with CleanResin; (6) transfer products of (5) to SpectroCHIP; and(7) analysis of the SpectroCHIP. In some of these embodiments, theamplification method is polymerase chain reaction, self-sustainedsequence reaction, ligase chain reaction, rapid amplification of cDNAends, polymerase chain reaction and ligase chain reaction, Q-beta phageamplification, strand displacement amplification, or splice overlapextension polymerase chain reaction. In some embodiments, the method ofamplification is by PCR. In some embodiments, the dephosphorylationreaction is catalyzed by shrimp alkaline phosphatase. In someembodiments, hybridization of primer is adjacent to the locus ofinterest. In some embodiments, the incorporated nucleotide is adideoxynucleotide or deoxynucleotide. In some embodiments, theincorporated nucleotide is labeled with radioactive molecule,fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate,biotin, derivative of biotin, phosphorescent moiety, luminescent moiety,electrochemiluminescent moiety, chromatic moiety, and moiety having adetectable electron spin resonance, electrical capacitance, dielectricconstant or electrical conductivity. In some embodiments, the labelednucleotide is labeled with a fluorescent molecule. In some embodiments,an agent that inhibits cell lysis has been added to the sample toinhibit the lysis of cells, if present, where the agent is membranestabilizer, cross-linker, or cell lysis inhibitor. In some embodiments,the agent is a cell lysis inhibitor. In some of these embodiments, thecell lysis inhibitor is formalin at a percentage selected from the groupconsisting of: 0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%,0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or2-3%. In some embodiments, the concentration of formalin in the sampleis 0.1%.

In some embodiments, the sequence of a locus of interest is determinedby (1) amplification of the locus of interest; (2) exonuclease treatmentof the products of (1); (3) single stranded DNA of (2) is annealed to anoligonucleotide; (4) incorporation of a nucleotide using the annealedtemplate and primer of (3); (5) detection of the incorporatednucleotide. In some embodiments, the amplification method is bypolymerase chain reaction, self-sustained sequence reaction, ligasechain reaction, rapid amplification of cDNA ends, polymerase chainreaction and ligase chain reaction, Q-beta phage amplification, stranddisplacement amplification, or splice overlap extension polymerase chainreaction. In some embodiments, the method of amplification is by PCR. Insome embodiments, the primer hybridizes adjacent to the locus ofinterest. In some embodiment, the incorporated nucleotide is adideoxynucleotide or deoxynucleotide. In some embodiments, theincorporation reaction comprises two terminating nucleotides and twonon-terminating nucleotides. In some embodiments, the incorporatednucleotide is labeled with radioactive molecule, fluorescent molecule,antibody, antibody fragment, hapten, carbohydrate, biotin, derivative ofbiotin, phosphorescent moiety, luminescent moiety,electrochemiluminescent moiety, chromatic moiety, and moiety having adetectable electron spin resonance, electrical capacitance, dielectricconstant or electrical conductivity. Income embodiments, the terminatingnucleotides are labeled with radioactive molecule, fluorescent molecule,antibody, antibody fragment, hapten, carbohydrate, biotin, derivative ofbiotin, phosphorescent moiety, luminescent moiety,electrochemiluminescent moiety, chromatic moiety, and moiety having adetectable electron spin resonance, electrical capacitance, dielectricconstant or electrical conductivity. In some embodiments, the labelednucleotide is labeled with a fluorescent molecule. In some embodiments,the terminating nucleotides are labeled with a fluorescent molecule. Insome embodiments, an agent that inhibits cell lysis has been added tothe sample to inhibit the lysis of cells, if present, where the agent ismembrane stabilizer, cross-linker, or cell lysis inhibitor. In someembodiments, the agent is a cell lysis inhibitor. In some of theseembodiments, the cell lysis inhibitor is formalin at a percentageselected from the group consisting of: 0.0001-0.03%, 0.03-0.05%,0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 09-1.2%,1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration offormalin in the sample is 0.1%.

In some embodiments, the sequence of a locus of interest is determinedby (1) amplification of the locus of interest, wherein the amplificationreaction comprises a forward primer, a reverse primer, and a probe thatanneals to the locus of interest, which is within the region of theamplicon; and (2) detection of the PCR products, wherein the amount ofPCR product is used to determine the presence or absence of a specificgenetic sequence. In some embodiments, the amplification is by PCR. Insome embodiments, the probe contains a reporter dye at the 5′ end andthe 3′ end contains a quenching dye. In some embodiments, the PCRproducts are detected using the ABI 7700 Sequence Detection System. Insome embodiments, an agent that inhibits cell lysis has been added tothe sample to inhibit the lysis of cells, if present, where the agent ismembrane stabilizer, cross-linker, or cell lysis inhibitor. In someembodiments, the agent is a cell lysis inhibitor. In some of theseembodiments, the cell lysis inhibitor is formalin at a percentageselected from the group consisting of: 0.0001-0.03%, 0.03-0.05%,0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.7%, 0.7-0.9%, 0.9-1.2%,1.2-1.5%, 1.5-2%, or 2-3%. In some embodiments, the concentration offormalin in the sample is 0.1%.

In another aspect, the invention provides methods for determining thesequence of a locus of interest in a sample containing fetal DNA.

In some embodiments, the method for determining the sequence includes(a) amplifying a locus of interest on a template DNA using a first andsecond primers, where the second primer contains a recognition site fora restriction enzyme such that digestion with the restriction enzymegenerates a 5′ overhang containing the locus of interest; (b) digestingthe amplified DNA with the restriction enzyme that recognizes therecognition site on the second primer; (c) incorporating a nucleotideinto the digested DNA of (b) by using the 5′ overhang containing thelocus of interest as a template; and (d) determining the sequence of thelocus of interest by determining the sequence of the DNA of (c).

In other embodiments, the method for determining the sequence includes(a) amplifying alleles of a locus of interest on a template DNA using afirst and second primers, where the second primer contains a recognitionsite for a restriction enzyme such that digestion with the restrictionenzyme generates a 5′ overhang containing the locus of interest; (b)digesting the amplified DNA with the restriction enzyme that recognizesthe recognition site on the second primer; (c) incorporating nucleotidesinto the digested DNA of (b), where a nucleotide that terminateselongation, and is complementary to the locus of interest of an allele,is incorporated into the 5′ overhang of said allele, and a nucleotidecomplementary to the locus of interest of a different allele isincorporated into the 5′ overhang of said different allele, and saidterminating nucleotide, which is complementary to a nucleotide in the 5′overhang of said different allele, is incorporated into the 5′ overhangof said different allele; and (d) determining the sequence of thealleles of a locus of interest by determining the sequence of the DNA of(c).

In embodiments, the sample is cell, tissue, blood, serum, plasma,saliva, urine, tears, vaginal secretion, sweat, umbilical cord blood,chorionic villi, amniotic fluid, embryonic tissue, embryo, a two-celledembryo, a four-celled embryo, an eight-celled embryo, a 16-celledembryo, a 32-celled embryo, a 64-celled embryo, a 128-celled embryo, a256-celled embryo, a 512-celled embryo, a 1024-celled embryo, lymphfluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, asciticfluid, fecal matter, or body exudates.

In another aspect, the invention provides methods for preparing a samplefor analysis that include isolating free nucleic acid from a sample thatcontains nucleic acid, where an agent that inhibits cell lysis has beenadded to the sample to inhibit lysis of cells, if cells are present,where the agent is membrane stabilizer, cross-linker, or cell lysisinhibitor. In this aspect, the portion of the sample that is to beanalyzed is the free nucleic acid, not the cellular portion. In anembodiment, the present invention provides a method for isolatingnucleic acid said method comprising (a) obtaining a sample containingnucleic acid; (b) adding a cell lysis inhibitor, cell membranestabilizer, or cross-linker to the sample of (a); and (c) isolatingnucleic acid. In an embodiment, the method is used for isolating freenucleic acid. In an embodiment, the method is used for isolating freefetal nucleic acid. In another embodiment, the present inventionprovides a method for isolating free fetal nucleic acid said methodcomprising (a) obtaining a sample containing nucleic acid; (b) adding acell lysis inhibitor, cell membrane stabilizer, or cross-linker to thesample of (a); (c) isolating the plasma from the blood sample, whereinthe plasma is isolated by centrifuging the blood sample; and (d)removing the supernatant, which contains the plasma, using procedures tominimize disruption of the “buffy-coat.”

In some embodiments, the agent is cell lysis inhibitor, and in some ofthese embodiments, the cell lysis inhibitor is glutaraldehyde,derivatives of glutaraldehyde, formaldehyde, formalin, and derivativesof formaldehyde, crosslinkers, primary amine reactive crosslinkers,sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfidereduction, carbohydrate reactive crosslinkers, carboxyl reactivecrosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP,APG, BASED, BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES,DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS,HBVS, sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS or compounds listed in TableXXIII.

In some embodiments the cell lysis inhibitor is formalin. In some ofthese embodiments, the final concentration of formalin in the sample is0.0001-0.03%, 0.03-0.05%, 0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%,0.5-0.7%, 0.7-0.9%, 0.9-1.2%, 1.2-1.5%, 1.5-2%, or 2-3%. In oneembodiment, the final concentration of formalin in the sample is 0.1%.

An agent that stabilizes cell membranes may be added to the sampleincluding but not limited to aldehydes, urea formaldehyde, phenolformaldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterolderivatives, high concentrations of magnesium, vitamin E, and vitamin Ederivatives, calcium, calcium gluconate, taurine, niacin, hydroxylaminederivatives, bimoclomol, sucrose, astaxanthin, glucose, amitriptyline,isomer A hopane tetral phenylacetate, isomer B hopane tetralphenylacetate, citicoline, inositol, vitamin B, vitamin B complex,cholesterol hemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone,vitamin K, vitamin K complex, menaquinone, zonegran, zinc, ginkgo bilobaextract, diphenylhydantoin, perftoran, polyvinylpyrrolidone,phosphatidylserine, tegretol, PABA, disodium cromglycate, nedocromilsodium, phenyloin, zinc citrate, mexitil, dilantin, sodium hyaluronate,or polaxamer 188.

In another embodiment, an agent that prevents DNA destruction is addedto the sample including but not limited to DNase inhibitors, zincchloride, ethylenediaminetetraacetic acid, guanidine-HCl, guanidineisothiocyanate, N-lauroylsarcosine, and Na-dodecylsulphate.

In some embodiments, the sample is obtained from human, non-human,mammal, reptile, cattle, cat, dog, goat, swine, pig, monkey, ape,gorilla, bull, cow, bear, horse, sheep, poultry, mouse, rat, fish,dolphin, whale, or shark. In some of these embodiments, the sample isobtained from a human source.

In some embodiments, the sample containing nucleic acid is obtained fromany nucleic acid containing source including but not limited to a cell,fetal cell, tissue, blood, serum, plasma, saliva, urine, tear, vaginalsecretion, breast fluid, breast milk, sweat, umbilical cord blood,chorionic villi, amniotic fluid, embryonic tissue, embryo, a two-celledembryo, a four-celled embryo, an eight-celled embryo, a 16-celledembryo, a 32-celled embryo, a 64-celled embryo, a 128-celled embryo, a256-celled embryo, a 512-celled embryo, a 1024-celled embryo lymphfluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, asciticfluid, fecal matter, or body exudates. In some of these embodiments, thesample is blood.

In embodiments the sample is from a pregnant female. In an embodiment,the sample is obtained from a pregnant human female. In an embodiment,the sample is blood obtained from a pregnant female and, e.g., thenucleic acid is isolated from plasma obtained from blood of a pregnantfemale; the plasma is generated using procedures designed to minimizethe amount of maternal cell lysis. In some of these embodiments, theblood is obtained from a human pregnant female when the fetus is at agestational age of 0-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32,32-36, 36-40, 40-44, 44-48, 48-52, or more than 52 weeks. In some ofthese embodiments, the sample is obtained from plasma from the blood.

In some embodiments, the isolation of nucleic acid includes acentrifugation step; e.g., in some embodiments free nucleic acid isisolated from plasma obtained from blood, for example from a pregnantfemale. In some embodiments, the centrifugation step is performed withthe centrifuge braking power set to zero (the centrifuge comes to a stopby natural deceleration). In some embodiments, the centrifugation stepis performed at a speed of 0-50 rpm, 50-100 rpm, 100-200 rpm, 200-300rpm, 300-400 rpm, 400-500 rpm, 500-600 rpm, 600-700 rpm, 700-800 rpm,800-900 rpm, 900-1000 rpm, 1000-2000 rpm, 2000-3000 rpm, 3000-4000 rpm,4000-5000 rpm, 5000-6000 rpm, 6000-7000 rpm, 7000-8000 rpm, or greaterthan 8000 rpm. In one embodiment, the blood, e.g., from the pregnantfemale, is centrifuged at a speed less than 4000 rpm. In anotherembodiment, the acceleration power of the centrifuge is not used.

In another aspect, the invention provides a method for detecting achromosomal abnormality by (a) determining the sequence of alleles of alocus of interest from template DNA, and (b) quantitating the relativeamount of the alleles at a heterozygous locus of interest that wasidentified from the locus of interest of (a), wherein said relativeamount is expressed as a ratio, and wherein said ratio indicates thepresence or absence of a chromosomal abnormality.

In yet another aspect, the invention provides compositions,

In one embodiment, the invention provides a composition containing fetalDNA and maternal DNA, where the percentage of free fetal DNA in thetotal free DNA of the composition is about 15-16% fetal DNA, about16-17% fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about19-20% fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about22-23% fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about25-35% fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about55-65% fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about85-90% fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about92-93% fetal DNA, about 93-94% fetal DNA, about 94-95% fetal DNA, about95-96% fetal DNA, about 96-97% fetal DNA, about 97-98% fetal DNA, about98-99% fetal DNA, or about 99-99.7% fetal DNA.

In another embodiment, the invention provides a composition containingfetal DNA and maternal DNA, where the percentage of free fetal DNA inthe total free DNA of the composition is about 15-16% fetal DNA, about16-17% fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about19-20% fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about22-23% fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about25-35% fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about55-65% fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about85-90% fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about92-93% fetal DNA, about 93-94% fetal DNA, or about 94-95% fetal DNA.

In yet another aspect, the invention provides a prenatal diagnosticmethod including analyzing a composition comprising fetal DNA andmaternal DNA, where the percentage of free fetal DNA in the total freeDNA of the composition is about 15-16% fetal DNA, about 16-17% fetalDNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about 19-20% fetalDNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about 22-23% fetalDNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about 25-35% fetalDNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about 55-65% fetalDNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about 85-90% fetalDNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about 92-93% fetalDNA, about 93-94% fetal DNA, or about 94-95% fetal DNA.

In still yet another aspect, the invention provides a kit for use in anyof the methods of the invention, where the kit contains a set of primersused in the method, where the second primer contains a sequence thatgenerates a recognition site for a restriction enzyme such thatdigestion with the restriction enzyme generates a 5′ overhang containingthe locus of interest, and a set of instructions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. A schematic diagram depicting a double stranded DNA molecule. Apair of primers, depicted as bent arrows, flank the locus of interest,depicted as a triangle symbol at base N14. The locus of interest can bea single nucleotide polymorphism, point mutation, insertion, deletion,translocation, etc. Each primer contains a restriction enzymerecognition site about 10 by from the 5′ terminus depicted as region “a”in the first primer and as region “d” in the second primer, Restrictionrecognition site “a” can be for any type of restriction enzyme butrecognition site “d” is for a restriction enzyme, which cuts “n”nucleotides away from its recognition site and leaves a 5′ overhang anda recessed 3′ end. Examples of such enzymes include but are not limitedto BceAI and BsmF I. The 5′ overhang serves as a template forincorporation of a nucleotide into the 3′ recessed end.

The first primer is shown modified with biotin at the 5′ end to aid inpurification. The sequence of the 3′ end of the primers is such that theprimers anneal at a desired distance upstream and downstream of thelocus of interest. The second primer anneals close to the locus ofinterest; the annealing site, which is depicted as region “c,” isdesigned such that the 3′ end of the second primer anneals one base awayfrom the locus of interest. The second primer can anneal any distancefrom the locus of interest provided that digestion with the restrictionenzyme, which recognizes the region “d” on this primer, generates a 5′overhang that contains the locus of interest. The first primer annealingsite, which is depicted as region “b,” is about 20 bases.

FIG. 1B. A schematic diagram depicting the annealing and extension stepsof the first cycle of amplification by PCR. The first cycle ofamplification is performed at about the melting temperature of the 3′region, which anneals to the template DNA, of the second primer,depicted as region “c,” and is 13 base pairs in this example. At thistemperature, both the first and second primers anneal to theirrespective complementary strands and begin extension, depicted by dottedlines. In this first cycle, the second primer extends and copies theregion b where the first primer can anneal in the next cycle.

FIG. 1C. A schematic diagram depicting the annealing and extension stepsfollowing denaturation in the second cycle of amplification of PCR. Thesecond cycle of amplification is performed at a higher annealingtemperature (TM2), which is about the melting temperature of the 20 byof the 3′ region of the first primer that anneals to the template DNA,depicted as region “b.” Therefore at TM2, the first primer, whichcontains region b′ which is complementary to region b, can bind to theDNA that was copied in the first cycle of the reaction. However, at TM2the second primer cannot anneal to the original template DNA or to DNAthat was copied in the first cycle of the reaction because the annealingtemperature is too high. The second primer can anneal to 13 bases in theoriginal template DNA but TM2 is calculated at about the meltingtemperature of 20 bases.

FIG. 1D. A schematic diagram depicting the annealing and extensionreactions after denaturation during the third cycle of amplification. Inthis cycle, the annealing temperature, TM3, is about the meltingtemperature of the entire second primer, including regions “c” and “d.”The length of regions “c”+“d” is about 27-33 by long, and thus TM3 issignificantly higher than TM1 and TM2. At this higher TM the secondprimer, which contain regions c′ and d′, anneals to the copied DNAgenerated in cycle 2.

FIG. 1E. A schematic diagram depicting the annealing and extensionreactions for the remaining cycles of amplification. The annealingtemperature for the remaining cycles is TM3, which is about the meltingtemperature of the entire second primer. At TM3, the second primer bindsto templates that contain regions c′ and d′ and the first primer bindsto templates that contain regions a′ and b. By raising the annealingtemperature successively in each cycle for the first three cycles, fromTM1, TM2, and TM3, nonspecific amplification is significantly reduced.

FIG. 1F. A schematic diagram depicting the amplified locus of interestbound to a solid matrix.

FIG. 1G. A schematic diagram depicting the bound, amplified. DNA afterdigestion with restriction enzyme “d.” The “downstream” end is releasedinto the supernatant, and can be removed by washing with any suitablebuffer. The upstream end containing the locus of interest remains boundto the solid matrix.

FIG. 1H. A schematic diagram depicting the bound amplified DNA, after“filling in” with a labeled ddNTP. A DNA polymerase is used to “fill in”the base (N′14) that is complementary to the locus of interest (N14). Inthis example, only ddNTPs are present in this reaction, such that onlythe locus of interest or SNP of interest is filled in.

FIG. 1I. A schematic diagram depicting the labeled, bound DNA afterdigestion with restriction enzyme “a.” The labeled DNA is released intothe supernatant, which can be collected to identify the base that wasincorporated.

FIG. 2. A schematic diagram depicting double stranded DNA templates withn number of loci of interest and n number of primer pairs, x₁, y₁ tox_(n), y_(n), specifically annealed such that a primer flanks each locusof interest. The first primers are biotinylated at the 5′ end, depictedby *, and contain a restriction enzyme recognition site, “a”, which canbe any type of restriction enzyme. The second primers contain arestriction enzyme recognition site, “d,” where “d” is a recognitionsite for a restriction enzyme that cuts “n” nucleotides away from itsrecognition site, and generates a 5′ overhang containing the locus ofinterest and a recessed 3′ end. The second primers anneal adjacent tothe respective loci of interest. The exact position of the restrictionenzyme site “d” in the second primers is designed such that digestingthe PCR product of each locus of interest with restriction enzyme “d”generates a 5′ overhang containing the locus of interest and a 3′recessed end. The annealing sites of the first primers are about 20bases long and are selected such that each successive first primer isfurther away from its respective second primer. For example, if at locus1 the 3′ ends of the first and second primers are Z base pairs apart,then at locus 2, the 3′ ends of the first and second primers are Z+Kbase pairs apart, where K=1, 2, 3 or more than three bases. Primers forlocus N are Z_(N-1)+K base pairs apart. The purpose of making eachsuccessive first primer further apart from their respective secondprimers is such that the “filled in” restriction fragments (generatedafter amplification, purification, digestion and labeling as describedin FIGS. 1B-1I) differ in size and can be resolved, for example byelectrophoresis, to allow detection of each individual locus ofinterest.

FIG. 3A-3C: PCR amplification of SNPs using multiple annealingtemperatures. A sample containing genomic DNA templates from thirty-sixhuman volunteers was analyzed for the following four SNPs: SNPHC21S00340 (lane 1), identification number as assigned in the HumanChromosome 21 cSNP Database, located on chromosome 21; SNP TSC 0095512(lane 2), located on chromosome 1, SNP TSC 0214366 (lane 3), located onchromosome 1; and SNP TSC 0087315 (lane 4), located on chromosome 1.Each SNP was amplified by PCR using three different annealingtemperature protocols, herein referred to as the low stringencyannealing temperature; medium stringency annealing temperature; and highstringency annealing temperature. Regardless of the annealingtemperature protocol, each SNP was amplified for 40 cycles of PCR. Thedenaturation step for each PCR reaction was performed for 30 seconds at95° C. 3A. Photograph of a gel demonstrating PCR amplification of the 4different SNPs using the low stringency annealing temperature protocol.3B. Photograph of a gel demonstrating PCR amplification of the 4different SNPs using medium stringency annealing temperature protocol.3C. Photograph of a gel demonstrating PCR amplification of the 4different SNPs using the high stringency annealing temperature protocol.

FIG. 4A. (From top to bottom: SEQ ID NOS: 17, 667, 668, 18.) A depictionof the DNA sequence of SNP HC21S00027, as assigned by the HumanChromosome 21 cSNP database, located on chromosome 21. A first primerand a second primer are indicated above and below, respectively, thesequence of HC21S00027. The first primer is biotinylated and containsthe restriction enzyme recognition site for EcoRI. The second primercontains the restriction enzyme recognition site for BsmF I and contains13 bases that anneal to the DNA sequence. The SNP is indicated by R(A/G) and r (T/C) (complementary to R).

FIG. 4B. (From top to bottom: SEQ ID NOS: 17, 667, 668, 19.) A depictionof the DNA sequence of SNP HC21S00027, as assigned by the HumanChromosome 21 cSNP database, located on chromosome 21. A first primerand a second primer are indicated above and below, respectively, thesequence of HC21S00027. The first primer is biotinylated and containsthe restriction enzyme recognition site for EcoRI. The second primercontains the restriction enzyme recognition site for BceA I and has 13bases that anneal to the DNA sequence. The SNP is indicated by R (A/G)and r (T/C) (complementary to R).

FIG. 4C. (From top to bottom: SEQ ID NOS: 11, 669, 670, 20.) A depictionof the DNA sequence of SNP TSC0095512 from chromosome 1. The firstprimer and the second primer are indicated above and below,respectively, the sequence of TSC0095512. The first primer isbiotinylated and contains the restriction enzyme recognition site forEcoRI. The second primer contains the restriction enzyme recognitionsite for BsmF I and has 13 bases that anneal to the DNA sequence. TheSNP is indicated by S (G/C) and s (C/G) (complementary to S).

FIG. 4D. (From top to bottom: SEQ ID NOS: 11, 669, 670, 12.) A depictionof the DNA sequence of SNP TSC0095512 from chromosome 1. The firstprimer and the second primer are indicated above and below,respectively, the sequence of TSC0095512. The first primer isbiotinylated and contains the restriction enzyme recognition site forEcoRI. The second primer contains the restriction enzyme recognitionsite for BceA I and has 13 bases that anneal to the DNA sequence. TheSNP is indicated by S (G/C) and s (C/G) (complementary to S).

FIGS. 5A-5D. (FIG. 5A: SEQ ID NOS: 671 (top) and 672 (bottom); FIG. 5B:SEQ ID NOS: 673 (top) and 674 (bottom); FIG. 5C: SEQ ID NOS: 675 (top)and 676 (bottom); FIG. 5D: SEQ ID NOS: 677 (top) and 678 (bottom)). Aschematic diagram depicting the nucleotide sequences of SNP HC21S00027(FIGS. 5A and 5B) and SNP TSC0095512 (FIGS. 5C and 5D) afteramplification with the primers described in FIGS. 4A-4D. Restrictionsites in the primer sequence are indicated in bold.

FIGS. 6A-6D. A schematic diagram depicting the nucleotide sequences ofeach amplified SNP after digestion with the appropriate Type IISrestriction enzyme. FIGS. 6A (SEQ ID NOS: 679 (upper left), 680 (upperright), 681 (lower left) and 682 (lower right)) and 6B (SEQ ID NOS: 679(upper left), 683 (upper right), 684 (lower left) and 685 (lower right))depict fragments of SNP HC21S00027 digested with the Type IISrestriction enzymes BsmF I and BceA I, respectively. FIGS. 6C (SEQ IDNOS: 686 (upper left), 687 (upper right), 688 (lower left) and 689(lower right)) and 6D (SEQ ID NOS: 686 (upper left), 690 (upper right),691 (lower left) and 692 (lower right)) depict fragments of SNPTSC0095512 digested with the Type IIS restriction enzymes BsmF I andBceA I, respectively.

FIGS. 7A-7D. A schematic diagram depicting the incorporation of afluorescently labeled nucleotide using the 5′ overhang of the digestedSNP site as a template to “fill in” the 3′ recessed end. FIGS. 7A (SEQID NOS: 693 (top) and 694 (bottom)) and 7B (SEQ ID NOS: 693 (top) and695 (bottom)) depict the digested SNP HC21S00027 locus with anincorporated labeled ddNTP (*R^(−dd)=fluorescent dideoxy nucleotide).FIGS. 7C (SEQ ID NOS: 696 (top) and 697 (bottom)) and 7D (SEQ ID NOS:696 (top) and 698 (bottom)) depict the digested SNP TSC0095512 locuswith an incorporated labeled ddNTP (*S^(−dd)=fluorescent dideoxynucleotide). The use of ddNTPs ensures that the 3′ recessed end isextended by one nucleotide, which is complementary to the nucleotide ofinterest or SNP site present in the 5′ overhang.

FIG. 7E. (From top to bottom: SEQ ID NOS: 693, 694, 699, 694, 700, 694,701, 694.) A schematic diagram depicting the incorporation of dNTPs anda ddNTP into the 5′ overhang containing the SNP site. SNP HC21500007 wasdigested with BsmF I, which generates a four base 5′ overhang. The useof a mixture of dNTPs and ddNTPs allows the 3′ recessed end to beextended one nucleotide (a ddNTP is incorporated first); two nucleotides(a dNTP is incorporated followed by a ddNTP); three nucleotides (twodNTPs are incorporated, followed by a ddNTP); or four nucleotides (threedNTPs are incorporated, followed by a ddNTP). All four products can beseparated by size, and the incorporated nucleotide detected(*R^(−dd)=fluorescent dideoxy nucleotide). Detection of the firstnucleotide, which corresponds to the SNP or locus site, and the nextthree nucleotides provides an additional level of quality assurance. TheSNP is indicated by R (A/G) and r (T/C) (complementary to R).

FIGS. 8A-8D. Release of the “filled in” SNP from the solid supportmatrix, i.e. streptavidin coated well. SNP HC21S00027 is shown in FIGS.8A (SEQ ID NOS: 702 (upper left), 703 (upper right), 704 (lower left)and 705 (lower right)) and 8B (SEQ ID NOS: 702 (upper left), 703 (upperright), 704 (lower left) and 706 (lower right)), while SNP TSC0095512 isshown in FIGS. 8C (SEQ ID NOS: 707 (upper left), 708 (upper right), 709(lower left) and 710 (lower right)) and 8D (SEQ ID NOS: 707 (upperleft), 708 (upper right), 709 (lower left) and 711 (lower right)). The“filled in” SNP is free in solution, and can be detected.

FIG. 9A. Sequence analysis of SNP HC21S00027 digested with BceAI. Four“fill in” reactions are shown; each reaction contained one fluorescentlylabeled nucleotide, ddGTP, ddATP, ddTTP, or ddCTP, and unlabeled ddNTPs.The 5′ overhang generated by digestion with BceA I and the expectednucleotides at this SNP site are indicated.

FIG. 9B. Sequence analysis of SNP TSC0095512. SNP TSC0095512 wasamplified with a second primer that contained the recognition site forBceA I, and in a separate reaction, with a second primer that containedthe recognition site for BsmF I. Four fill in reactions are shown foreach PCR product; each reaction contained one fluorescently labelednucleotide, ddGTP, ddATP, ddTTP, or ddCTP, and unlabeled ddNTPs. The 5′overhang generated by digestion with BceA I and with BsmF I and theexpected nucleotides are indicated.

FIG. 9C. Sequence analysis of SNP TSC0264580 after amplification with asecond primer that contained the recognition site for BsmF I. Four fillin reactions are shown; each reaction contained one fluorescentlylabeled nucleotide, which was ddGTP, ddATP, ddTTP, or ddCTP andunlabeled ddNTPs. Two different 5′ overhangs are depicted: onerepresents the DNA molecules that were cut 11 nucleotides away on thesense strand and 15 nucleotides away on the antisense strand and theother represents the DNA molecules that were cut 10 nucleotides away onthe sense strand and 14 nucleotides away on the antisense strand. Theexpected nucleotides also are indicated.

FIG. 9D. Sequence analysis of SNP HC21 S00027 amplified with a secondprimer that contained the recognition site for BsmF I. A mixture oflabeled ddNTPs and unlabeled dNTPs was used to fill in the 5′ overhanggenerated by digestion with BsmF I. Two different 5′ overhangs aredepicted: one represents the DNA molecules that were cut 11 nucleotidesaway on the sense strand and 15 nucleotides away on the antisense strandand the other represents the DNA molecules that were cut 10 nucleotidesaway on the sense strand and 14 nucleotides away on the antisensestrand. The nucleotide upstream from the SNP, the nucleotide at the SNPsite (the sample contained DNA templates from 36 individuals; bothnucleotides would be expected to be represented in the sample), and thethree nucleotides downstream of the SNP are indicated.

FIG. 10. Sequence analysis of multiple SNPs. SNPs HC21S00131, andHC21S00027, which are located on chromosome 21, and SNPs TSC0087315, SNPTSC0214366, SNP TSC0413944, and SNP TSC0095512, which are on chromosome1, were amplified in separate PCR reactions with second primers thatcontained a recognition site for BsmF I. The primers were designed sothat each amplified locus of interest was of a different size. Afteramplification, the reactions were pooled into a single sample, and allsubsequent steps of the method performed (as described for FIGS. 1F-1I)on that sample. Each SNP and the nucleotide found at each SNP areindicated.

FIG. 11A-11B. Quantification of the percentage of fetal DNA in maternalblood. Blood was obtained from a pregnant human female with informedconsent. DNA was isolated and serial dilutions were made to determinethe percentage of fetal DNA present in the sample. The SRY gene, whichis located on chromosome Y, was used to detect fetal DNA. The cysticfibrosis gene, which is located on chromosome 7, was used to detect bothmaternal and fetal DNA. 11 A. Amplification of the SRY gene and thecystic fibrosis gene using a DNA template isolated from a blood samplethat was treated with EDTA. 11B. Amplification of the SRY gene and thecystic fibrosis gene using a DNA template that was isolated from a bloodsample that was treated with formalin and EDTA.

FIG. 12. Genetic analysis of an individual previously genotyped withTrisomy 21 (Down's Syndrome). Blood was collected, with informedconsent, from an individual who had previously been genotyped withtrisomy 21. DNA was isolated and two SNPs on chromosome 21 and two SNPson chromosome 13 were genotyped. As shown in the photograph of the gel,the SNPs at chromosome 21 show disproportionate ratios of the twonucleotides. Visual inspection of the gel demonstrates that onenucleotide of the two nucleotides at the SNP sites analyzed forchromosome 21 is of greater intensity, suggesting it is not present in a50:50 ratio. However, visual inspection of the gel suggests that thenucleotides at the heterozygous SNP sites analyzed on chromosome 13 arepresent in the expected 50:50 ratio.

FIG. 13. Sequence determination of both alleles of SNPs TSC0837969,TSC0034767, TSC1130902, TSC0597888, TSC0195492, TSC0607185 using onefluorescently labeled nucleotide. Labeled ddGTP was used in the presenceof unlabeled dATP, dCTP, dTTP to fill-in the overhang generated bydigestion with BsmF I. The nucleotide preceding the variable site on thestrand that was filled-in was not guanine, and the nucleotide after thevariable site on the strand that was filled in was not guanine. Thenucleotide two bases after the variable site on the strand that wasfilled-in was guanine. Alleles that contain guanine at variable site arefilled in with labeled ddGTP. Alleles that do not contain guanine arefilled in with unlabeled dATP, dCTP, or dTTP, and the polymerasecontinues to incorporate nucleotides until labeled ddGTP is filled in atposition 3 complementary to the overhang.

FIG. 14. Identification of SNPs with alleles that are variable withinthe population. The sequences of both alleles of seven SNPs located onchromosome 13 were determined using a template DNA comprised of DNAobtained from two hundred and forty five individuals. Labeled ddGTP wasused in the presence of unlabeled dATP, dCTP, dTTP to fill-in theoverhang generated by digestion with BsmF I. The nucleotide precedingthe variable site on the strand that was filled-in was not guanine, andthe nucleotide after the variable site on the strand that was filled inwas not guanine. The nucleotide two bases after the variable site on thestrand that was filled-in was guanine. Alleles that contain guanine atvariable site are filled in with labeled ddGTP. Alleles that do notcontain guanine are filled in with unlabeled dATP, dCTP, or dTTP, andthe polymerase continues to incorporate nucleotides until labeled ddGTPis filled in at position 3 complementary to the overhang.

FIG. 15. Determination of the ratio for one allele to the other alleleat heterozygous SNPs. The observed nucleotides for SNP TSC0607185 arecytosine (referred to as allele 1) and thymidine (referred to as allele2) on the sense strand. The ratio of allele 2 to allele 1 was calculatedusing template DNA isolated from five individuals. The ratio of allele 2to allele 1 (allele 2/allele 1) was consistently 1:1.

The observed nucleotides for SNP TSC1130902 are guanine (referred to asallele 1) and adenine (referred to as allele 2) on the sense strand. Theratio of allele 2 to allele 1 was calculated using template DNA isolatedfrom five individuals. The ratio of allele 2 to allele 1 (allele2/allele 1) was consistently 75:25.

FIG. 16. The percentage of allele 2 to allele 1 at SNP TSC0108992remains linear when calculated on template DNA containing an extra copyof chromosome 21. SNP TSC0108992 was amplified using template DNA fromfour individuals, and two separate fill-in reactions (labeled as A andB) were performed for each PCR reaction (labeled 1 through 4). Thecalculated percentage of allele 2 to allele 1 on template DNA fromnormal individuals was 0.47. The deviation from the theoreticallypredicted percentage of 0.50 remained linear on template DNA isolatedfrom an individual with Down's syndrome.

FIG. 17A. Analysis of a SNP located on chromosome 21 from template DNAisolated from an individual with a normal genetic karyotype. SNPTSC0108992 was amplified using the methods described herein, and afterdigestion with the type IIS restriction enzyme BsmF I, the 5′ overhangwas filled in using labeled ddTTP, and unlabeled dATP, dCTP, and dGTP.Three separate PCR reactions were performed, and each PCR reaction wassplit into two samples. The percentage of allele 2 at the SNP site(allele 2/(allele 2+allele 1)) was calculated, which resulted in mean of0.50.

FIG. 17B. Analysis of a SNP located on chromosome 21 from template DNAisolated from an individual with a trisomy 21 genetic karyotype. SNPTSC0108992 was amplified using the methods described herein, and afterdigestion with the type IIS restriction enzyme BsmF I, the 5′ overhangwas filled in using labeled ddTTP, and unlabeled dATP, dCTP, and dGTP.Three separate PCR reactions were performed, and each PCR reaction wassplit into two samples. The percentage of allele 2 at the SNP site(allele 2/(allele 2+allele 1)) was calculated, which resulted in mean of0.30.

FIG. 17C. Analysis of a SNP located on chromosome 21 from a mixturecomprised of template DNA from an individual with Trisomy 21, andtemplate DNA from an individual with a normal genetic karyotype in aratio of 3:1 (Trisomy 21: Normal). SNP TSC0108992 was amplified from themixture of template DNA using the methods described herein, and afterdigestion with the type IIS restriction enzyme BsmF I, the 5′ overhangwas filled in using labeled ddTTP, and unlabeled dATP, dCTP, and dGTP.Three separate PCR reactions were performed, and each PCR reaction wassplit into two samples. The percentage of allele 2 at the SNP site(allele 2/(allele 2+allele 1)) was calculated, which resulted in mean of0.319.

FIG. 17D. Analysis of a SNP located on chromosome 21 from a mixturecomprised of template DNA from an individual with Trisomy 21, andtemplate DNA from an individual with a normal genetic karyotype in aratio of 1:1 (Trisomy 21: Normal). SNP TSC0108992 was amplified from themixture of template DNA using the methods described herein, and afterdigestion with the type IIS restriction enzyme BsmF I, the 5′ overhangwas filled in using labeled ddTTP, and unlabeled dATP, dCTP, and dGTP.Three separate PCR reactions were performed, and each PCR reaction wassplit into two samples. The percentage of allele 2 at the SNP site(allele 2/(allele 2+allele 1)) was calculated, which resulted in mean of0.352.

FIG. 17E. Analysis of a SNP located on chromosome 21 from a mixturecomprised of template DNA from an individual with Trisomy 21, andtemplate DNA from an individual with a normal genetic karyotype in aratio of 1:2.3 (Trisomy 21: Normal). SNP TSC0108992 was amplified fromthe mixture of template DNA using the methods described herein, andafter digestion with the type IIS restriction enzyme BsmF I, the 5′overhang was filled in using labeled ddTTP, and unlabeled dATP, dCTP,and dGTP. Three separate PCR reactions were performed, and each PCRreaction was split into two samples. The percentage of allele 2 at theSNP site (allele 2/(allele 2+allele 1)) was calculated, which resultedin mean of 0.382.

FIG. 17F. Analysis of a SNP located on chromosome 21 from a mixturecomprised of template DNA from an individual with Trisomy 21, andtemplate DNA from an individual with a normal genetic karyotype in aratio of 1:4 (Trisomy 21: Normal). SNP TSC0108992 was amplified from themixture of template DNA using the methods described herein, and afterdigestion with the type IIS restriction enzyme BsmF I, the 5′ overhangwas filled in using labeled ddTTP, and unlabeled dATP, dCTP, and dGTP.Three separate PCR reactions were performed, and each PCR reaction wassplit into two samples. The percentage of allele 2 at the SNP site(allele 2/(allele 2+allele 1)) was calculated, which resulted in mean of0.397.

FIG. 18A. Agarose gel analysis of nine (9) SNPs amplified from templateDNA. Each of the nine SNPs were amplified from genomic DNA using themethods described herein. Lane 1 corresponds to SNP TSC0397235, lane 2corresponds to TSC0470003, lane 3 corresponds to TSC1649726, lane 4corresponds to TSC1261039, lane 5 corresponds to TSC0310507, lane 6corresponds to TSC1650432, lane 7 corresponds to TSC1335008, lane 8corresponds to TSC0128307, and lane 9 corresponds to TSC0259757.

FIG. 18B. The original template DNA was amplified using 12 base primersthat annealed to various regions on chromosome 13. One hundred differentprimer sets were used to amplify regions throughout chromosome 13. Foreach of the nine SNPs, a primer that annealed approximately 130 basesfrom the locus of interest and 130 bases downstream of the locus ofinterest were used. This amplification reaction, which contained a totalof 100 different primer sets, was used to amplify the regions containingthe loci of interest. The resulting PCR product was used in a subsequentPCR reaction, wherein each of the nine SNPs were individually amplifiedusing a first primer and a second primer, wherein the second primercontained the binding site for the type IIs restriction enzyme BsmF I.SNPs were loaded in the same order as FIG. 18A.

FIG. 19A. Quantification of the percentage of allele 2 to allele 1 forSNP TSC047003 on original template DNA (IA) and multiplexed template DNA(M1-M3), wherein the DNA was first amplified using 12 base primers thatannealed 150 bases upstream and downstream of the loci of interest.Then, three separate PCR reactions were performed on the multiplexedtemplate DNA, using a first and second primer.

FIG. 19B. Quantification of the percentage of allele 2 to allele 1 forSNP TSC1261039 on original template DNA (IA) and multiplexed templateDNA (M1-M3), wherein the DNA was first amplified using 12 base primersthat annealed 150 bases upstream and downstream of the loci of interest.Then, three separate PCR reactions were performed on the multiplexedtemplate DNA, using a first and second primer.

FIG. 19C. Quantification of the percentage of allele 2 to allele 1 forSNP TSC310507 on original template DNA (IA) and multiplexed template DNA(M1-M3), wherein the DNA was first amplified using 12 base primers thatannealed 150 bases upstream and downstream of the loci of interest.Then, three separate PCR reactions were performed on the multiplexedtemplate DNA, using a first and second primer.

FIG. 19D. Quantification of the percentage of allele 2 to allele 1 forSNP TSC1335008 on original template DNA (IA) and multiplexed templateDNA (M1-M3), wherein the DNA was first amplified using 12 base primersthat annealed 150 bases upstream and downstream of the loci of interest.Then, three separate PCR reactions were performed on the multiplexedtemplate DNA, using a first and second primer.

FIG. 20. Detection of fetal DNA from plasma DNA isolated from a pregnantfemale. Four SNPs wherein the maternal DNA was homozygous were analyzedon the plasma. DNA. The maternal DNA was homozygous for adenine atTSC0838335 (lane 1), while the plasma DNA displayed a heterozygouspattern (lane 2). The guanine allele represented the fetal DNA, whichwas clearly distinguished from the maternal signal. Both the maternalDNA and the plasma DNA were homozygous for adenine at TSC0418134 (lanes3 and 4). The maternal DNA was homozygous for guanine at TSC0129188(lane 5), while the plasma DNA displayed a heterozygous pattern (lane6). The adenine allele represented the fetal DNA. Both the maternal DNAand the plasma DNA were homozygous for adenine at TSC0501389 (lanes 7and 8).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for detecting genetic disorders,including but not limited to mutations, insertions, deletions, andchromosomal abnormalities, and is especially useful for the detection ofgenetic disorders of a fetus. The method is especially useful fordetection of a translocation, addition, amplification, transversion,inversion, aneuploidy, polyploidy, monosomy, trisomy, trisomy 21,trisomy 13, trisomy 14, trisomy 15, trisomy 16, trisomy 18, trisomy 22,triploidy, tetraploidy, and sex chromosome abnormalities including butnot limited to XO, XXY, XYY, and XXX. The method also provides anon-invasive technique for determining the sequence of fetal DNA andidentifying mutations within the fetal DNA.

The invention is directed to a method for detecting chromosomalabnormalities, the method comprising: (a) determining the sequence ofalleles of a locus of interest on a template DNA; and (b) quantitating aratio for the alleles at a heterozygous locus of interest that wasidentified from the locus of interest of (a), wherein said ratioindicates the presence or absence of a chromosomal abnormality.

In another embodiment, the present invention provides a non-invasivemethod for determining the sequence of a locus of interest on fetal DNA,said method comprising: (a) obtaining a sample from a pregnant female;(b) adding a cell lysis inhibitor, cell membrane stabilizer orcross-linker to the sample of (a); (c) obtaining template DNA from thesample of (b), wherein said template DNA comprises fetal DNA andmaternal DNA; and (d) determining the sequence of a locus of interest ontemplate DNA.

In another embodiment, the present invention is directed to a method forisolating DNA, said method comprising (a) obtaining a sample containingnucleic acid; (b) adding a cell lysis inhibitor, cell membranestabilizer or cross-linker to sample of (a); and (c) isolating the DNA.

In another embodiment, the present invention is directed to a method forisolating free DNA, said method comprising (a) obtaining a samplecontaining nucleic acid; (b) adding a cell lysis inhibitor, cellmembrane stabilizer or cross-linker to sample of (a); and (c) isolatingthe DNA.

In another embodiment, the present invention is directed to a method forisolating free DNA from a sample containing nucleic acid to which a celllysis inhibitor, cell membrane stabilizer or cross-linker has beenadded, said method comprising isolating the DNA.

In another embodiment, the present invention is directed to a method forisolating free fetal DNA, said method comprising (a) obtaining a samplecontaining nucleic acid; (b) adding a cell lysis inhibitor, cellmembrane stabilizer or cross-linker to sample of (a); and (c) isolatingthe DNA. In another embodiment, the DNA is isolated using any techniquesuitable in the art including but not limited to cesium chloridegradients, gradients, sucrose gradients, glucose gradients,centrifugation protocols, boiling, Qiagen purification systems, QIA DNAblood purification kit, HiSpeed Plasmid Maxi Kit, QIAfilter plasmid kit,Promega DNA purification systems, MangeSil Paramagnetic Particle basedsystems, Wizard SV technology, Wizard Genomic DNA purification kit,Amersham purification systems, GFX Genomic Blood DNA purification kit,Invitrogen Life Technologies Purification Systems, CONCERT purificationsystem, Mo Bio Laboratories purification systems, UltraClean BloodSpinKits, and UlraClean Blood DNA Kit.

In another embodiment, the present invention is directed to a method forisolating free fetal DNA from a sample containing nucleic acid to whicha cell lysis inhibitor, cell membrane stabilizer or cross-linker hasbeen added, said method comprising isolating the DNA. In a preferredembodiment, the free fetal DNA is isolated from plasma or serum obtainedfrom the blood of a pregnant female.

In another embodiment, the DNA is isolated using techniques and/orprotocols that substantially reduce the amount of maternal DNA in thesample including but not limited to centrifuging the samples, with thebraking power for the centrifuge set to zero (the brake on thecentrifuge is not used), transferring the supernatant to a new tube withminimal or no disturbance of the “buffy-coat,” and transferring only aportion of the supernatant to a new tube. In a preferred embodiment,both acceleration power and braking power for the centrifuge are set tozero.

In another embodiment, the DNA is isolated using techniques and/orprotocols that substantially reduce the amount of maternal DNA in thesample including but not limited to centrifuging the samples, with theacceleration power for the centrifuge set to zero, transferring thesupernatant to a new tube with minimal or no disturbance of the“buffy-coat,” and transferring only a portion of the supernatant to anew tube.

In another embodiment, the “buffy-coat” is removed from the tube priorto removal of the supernatant using any applicable method including butnot limited to using a syringe or needle to withdraw the “buffy-coat,”

In another embodiment, the braking power for the centrifuge is set at apercentage including but not limited to 1-5%, 5-10%, 10-20%, 20-30%,30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99% ofmaximum braking power.

In another embodiment, the acceleration power for the centrifuge is setat a percentage including but not limited to 1-5%, 5-10%, 10-20%,20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%of maximum acceleration power.

In another embodiment, the present invention is directed to acomposition comprising free fetal DNA and free maternal DNA, wherein thecomposition comprises a relationship of free fetal DNA to free maternalDNA including but not limited to at least about 15% free fetal DNA, atleast about 20% free fetal DNA, at least about 30% free fetal DNA, atleast about 40% free fetal DNA, at least about 50% free fetal DNA, atleast about 60% free fetal DNA, at least about 70% free fetal DNA, atleast about 80% free fetal DNA, at least about 90% free fetal DNA, atleast about 91% free fetal DNA, at least about 92% free fetal DNA, atleast about 93% free fetal DNA, at least about 94% free fetal DNA, atleast about 95% free fetal DNA, at least about 96% free fetal DNA, atleast about 97% free fetal DNA, at least about 98% free fetal DNA, atleast about 99% free fetal DNA, and at least about 99.5% free fetal DNA.

In another embodiment, the present invention is directed to a method ofusing a composition comprising free fetal DNA and free maternal DNA forprenatal diagnostics, wherein the composition comprises a relationshipof free fetal DNA to free maternal DNA including but not limited to atleast about 15% free fetal DNA, at least about 20% free fetal DNA, atleast about 30% free fetal DNA, at least about 40% free fetal DNA, atleast about 50% free fetal DNA, at least about 60% free fetal DNA, atleast about 70% free fetal DNA, at least about 80% free fetal DNA, atleast about 90% free fetal DNA, at least about 91% free fetal DNA, atleast about 92% free fetal DNA, at least about 93% free fetal DNA, atleast about 94% free fetal DNA, at least about 95% free fetal DNA, atleast about 96% free fetal DNA, at least about 97% free fetal DNA, atleast about 98% free fetal. DNA, at least about 99% free fetal DNA, andat least about 99.5% free fetal DNA.

In another embodiment, the present invention is directed to acomposition comprising free fetal DNA and free maternal DNA, wherein thecomposition comprises a relationship of free fetal DNA to free maternalDNA including but not limited to about 13-15% free fetal DNA, about15-16% free fetal DNA, about 16-17% free fetal DNA, about 17-18% freefetal DNA, about 18-19% free fetal DNA, about 19-20% free fetal DNA,about 20-21% free fetal DNA, about 21-22% free fetal DNA, about 22-23%free fetal DNA, about 23-24% free fetal DNA, about 24-25% free fetalDNA, about 25-35% free fetal DNA, about 35-45% free fetal DNA, about45-55% free fetal DNA, about 55-65% free fetal DNA, about 65-75% freefetal DNA, about 75-85% free fetal DNA, about 85-90% free fetal DNA,about 90-91% free fetal DNA, about 91-92% free fetal DNA, about 92-93%free fetal DNA, about 93-94% free fetal DNA, about 94-95% free fetalDNA, about 95-96% free fetal DNA, about 96-97% free fetal DNA, about97-98% free fetal DNA, about 98-99% free fetal DNA, and about 99-99.7%free fetal DNA.

In another embodiment, the present invention is directed to a method ofusing a composition comprising free fetal DNA and free maternal DNA forprenatal diagnostics, wherein the composition comprises a relationshipof free fetal DNA to free maternal DNA including but not limited toabout 13-15% free fetal DNA, about 15-16% free fetal DNA, about 16-17%free fetal DNA, about 17-18% free fetal DNA, about 18-19% free fetalDNA, about 19-20% free fetal DNA, about 20-21% free fetal DNA, about21-22% free fetal DNA, about 22-23% free fetal DNA, about 23-24% freefetal DNA, about 24-25% free fetal DNA, about 25-35% free fetal DNA,about 35-45% free fetal DNA, about 45-55% free fetal DNA, about 55-65%free fetal DNA, about 65-75% free fetal DNA, about 75-85% free fetalDNA, about 85-90% free fetal DNA, about 90-91% free fetal DNA, about91-92% free fetal DNA, about 92-93% free fetal DNA, about 93-94% freefetal DNA, about 94-95% free fetal DNA, about 95-96% free fetal DNA,about 96-97% free fetal DNA, about 97-98% free fetal DNA, about 98-99%free fetal DNA, or about 99-99.7% free fetal DNA.

In another embodiment, the present invention is directed to acomposition comprising free fetal DNA and free maternal DNA, wherein thecomposition comprises a relationship of free fetal DNA to free maternalDNA including but not limited a maximum of 13%-15% free fetal DNA, amaximum of 15-18% free fetal DNA, a maximum of 18-20% free fetal DNA, amaximum of 20-40% free fetal DNA, a maximum of 40-50% free fetal DNA, amaximum of 50-60% free fetal DNA, a maximum of 60-70% free fetal DNA, amaximum of 70-80% free fetal DNA, a maximum of 80-90% free fetal DNA, amaximum of 90-92% free fetal DNA, a maximum of 92-94% free fetal DNA, amaximum of 94-95% free fetal DNA, a maximum of 95-96% free fetal DNA, amaximum of 96-97% free fetal DNA, a maximum of 97-98% free fetal DNA, amaximum of 98-99% free fetal DNA, a maximum of 99-99.5% free fetal DNA,and a maximum of 99.5-99.9% free fetal DNA.

In another embodiment, the present invention is directed to a method ofusing a composition comprising free fetal DNA and free maternal DNA forprenatal diagnostics, wherein the composition comprises a relationshipof free fetal DNA to free maternal DNA including but not limited amaximum of 13%-15% free fetal DNA, a maximum of 15-18% free fetal DNA, amaximum of 18-20% free fetal DNA, a maximum of 20-40% free fetal DNA, amaximum of 40-50% free fetal DNA, a maximum of 50-60% free fetal DNA, amaximum of 60-70% free fetal DNA, a maximum of 70-80% free fetal DNA, amaximum of 80-90% free fetal DNA, a maximum of 90-92% free fetal DNA, amaximum of 92-94% free fetal DNA, a maximum of 94-95% free fetal DNA, amaximum of 95-96% free fetal DNA, a maximum of 96-97% free fetal DNA, amaximum of 97-98% free fetal DNA, a maximum of 98-99% free fetal DNA, amaximum of 99-99.5% free fetal DNA, and a maximum of 99.5-99.9% freefetal DNA.

DNA Template

By a “locus of interest” is intended a selected region of nucleic acidthat is within a larger region of nucleic acid. A locus of interest caninclude but is not limited to 1-100, 1-50, 1-20, or 1-10 nucleotides,preferably 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotide(s).

As used herein, an “allele” is one of several alternate forms of a geneor non-coding regions of DNA that occupy the same position on achromosome. The term allele can be used to describe DNA from anyorganism including but not limited to bacteria, viruses, fungi,protozoa, molds, yeasts, plants, humans, non-humans, animals, andarcheabacteria.

For example, bacteria typically have one large strand of DNA. The termallele with respect to bacterial DNA refers to the form of a gene foundin one cell as compared to the form of the same gene in a differentbacterial cell of the same species.

Alleles can have the identical sequence or can vary by a singlenucleotide or more than one nucleotide. With regard to organisms thathave two copies of each chromosome, if both chromosomes have the sameallele, the condition is referred to as homozygous. If the alleles atthe two chromosomes are different, the condition is referred to asheterozygous. For example, if the locus of interest is SNP X onchromosome 1, and the maternal chromosome contains an adenine at SNP X(A allele) and the paternal chromosome contains a guanine at SNP X (Gallele), the individual is heterozygous at SNP X.

As used herein, sequence means the identity of one nucleotide or morethan one contiguous nucleotides in a polynucleotide. In the case of asingle nucleotide, e.g., a SNP, “sequence” and “identity” are usedinterchangeably herein.

The term “chromosomal abnormality” refers to a deviation between thestructure of the subject chromosome and a normal homologous chromosome.The term “normal” refers to the predominate karyotype or banding patternfound in healthy individuals of a particular species. A chromosomalabnormality can be numerical or structural, and includes but is notlimited to aneuploidy, polyploidy, inversion, a trisomy, a monosomy,duplication, deletion, deletion of a part of a chromosome, addition,addition of a part of chromosome, insertion, a fragment of a chromosome,a region of a chromosome, chromosomal rearrangement, and translocation.A chromosomal abnormality can be correlated with presence of apathological condition or with a predisposition to develop apathological condition. As defined herein, a single nucleotidepolymorphism (“SNP”) is not a chromosomal abnormality.

As used herein, incorporation of a nucleotide by a polymerase isreferred to as an elongation reaction or a fill-in reactioninterchangeably.

As used herein with respect to individuals, “mutant alleles” refers tovariant alleles that are associated with a disease state.

The term “template” refers to any nucleic acid molecule that can be usedfor amplification in the invention. RNA or DNA that is not naturallydouble stranded can be made into double stranded DNA so as to be used astemplate DNA. Any double stranded DNA or preparation containingmultiple, different double stranded DNA molecules can be used astemplate DNA to amplify a locus or loci of interest contained in thetemplate DNA.

The template DNA can be obtained from any source including but notlimited to humans, non-humans, mammals, reptiles, cattle, cats, dogs,goats, swine, pigs, monkeys, apes, gorillas, bulls, cows, bears, horses,sheep, poultry, mice, rats, fish, dolphins, whales, and sharks.

The template DNA can be from any appropriate sample including but notlimited to, nucleic acid-containing samples of tissue, bodily fluid (forexample, blood, serum, plasma, saliva, urine, tears, peritoneal fluid,ascitic fluid, vaginal secretion, breast fluid, breast milk, lymphfluid, cerebrospinal fluid or mucosa secretion), umbilical cord blood,chorionic villi, amniotic fluid, an embryo, a two-celled embryo, afour-celled embryo, an eight-celled embryo, a 16-celled embryo, a32-celled embryo, a 64-celled embryo, a 128-celled embryo, a 256-celledembryo, a 512-celled embryo, a 1024-celled embryo, embryonic tissues,lymph fluid, cerebrospinal fluid, mucosa secretion, or other bodyexudate, fecal matter, an individual cell or extract of the such sourcesthat contain the nucleic acid of the same, and subcellular structuressuch as mitochondria, using protocols well established within the art.

In one embodiment, the template DNA can be obtained from a sample of apregnant female.

In another embodiment, the template DNA can be obtained from an embryo.In a preferred embodiment, the template DNA can be obtained from asingle-cell of an embryo.

In one embodiment, the template DNA is fetal DNA. Fetal DNA can beobtained from sources including but not limited to maternal blood,maternal serum, maternal plasma, fetal cells, umbilical cord blood,chorionic villi, amniotic fluid, urine, saliva, cells or tissues.

In another embodiment, a cell lysis inhibitor is added to the sampleincluding but not limited to formaldehyde, formaldehyde derivatives,formalin, glutaraldehyde, glutaraldehyde derivatives, primary aminereactive crosslinkers, sulfhydryl reactive crosslinkers, sulfhydryladdition or disulfide reduction, carbohydrate reactive crosslinkers,carboxyl reactive crosslinkers, photoreactive crosslinkers, cleavablecrosslinkers, AEDP, APG, BASED, BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH,BMOE, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST,DTBP, DTME, DTSSP, EGS, HBVS, sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS orcompounds listed in Table XXIII. In another embodiment, two, three,four, five or more than five cell lysis inhibitors can be added to thesample. In a preferred embodiment, formalin is present in the sample ata percentage including but not limited to 0.0001-0.03%, 0.03-0.05%,0.05-0.08%, 0.08-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-0.3%, 0.7-0.9%, 0.9-1.2%,1.2-1.5%, 1.5-2%, 2-3%, 3-5%, and greater than 5%. In anotherembodiment, any combination of cross-linker, cell membrane stabilizer,or cell lysis inhibitor can be added to the sample including but notlimited to a cross-linker and a cell membrane stabilizer, a cross-linkerand a cell lysis inhibitor, and a cell membrane stabilizer and a celllysis inhibitor. More than one cross-linker can be used with more thanone cell membrane stabilizer. More than one cross-linker can be usedwith more than one cell lysis inhibitor. More than one cell membranestabilizer can be used with more than cell lysis inhibitor.

In another embodiment, the cell lysis inhibitor is added to the samplesuch that lysis is less than about 10% of the cells. In a preferredembodiment, the cell lysis inhibitor is added to the sample such thatlysis is less than about 5% of the cells. In a most preferredembodiment, the cell lysis inhibitor is added to the sample such thatlysis is less than about 1% of the cells.

In another embodiment, a cell membrane stabilizer is added to the samplesuch that lysis is less than about 10% of the cells. In a preferredembodiment, the cell membrane stabilizer is added to the sample suchthat lysis is less than about 5% of the cells. In a most preferredembodiment, the cell membrane stabilizer is added to the sample suchthat lysis is less than about 1% of the cells.

In another embodiment, a cross-linker is added to the sample such thatlysis is less than about 10% of the cells. In a preferred embodiment,the cross-linker is added to the sample such that lysis is less thanabout 5% of the cells. In a most preferred embodiment, the cross-linkeris added to the sample such that lysis is less than about 1% of thecells.

In another embodiment, the cell lysis inhibitor, cross-linker or cellmembrane stabilizer is added to the sample in an applicable time periodincluding but not limited to 1-10 seconds, 10-30 seconds, 30-60 seconds,1-5 minutes, 5-10 minutes, 10-20 minutes, 20-30 minutes, 30-40 minutes,40-50 minutes, 60-90 minutes, 90-180 minutes or greater than 180 minutesafter collection of the sample. In another embodiment, the cell lysisinhibitor, cross-linker, or cell membrane stabilizer is present in theapparatus to which the sample is collected including but not limited toa glass tube, a plastic tube, a circular container, an eppendorf tube,an IV bag, or any other appropriate collection device. In anotherembodiment, after the addition of the cell lysis inhibitor, cellmembrane stabilizer, or cross-linker, the sample is left at about roomtemperature for the period of time to allow the reagent to function,including but not limited to 1-5, 5-10, 10-20, 20-40, 40-60, 60-90,90-120, 120-150, 150-180, 180-240, 240-300 or greater than 300 minutes.

In another embodiment, the template DNA contains both maternal DNA andfetal DNA. In a preferred embodiment, template DNA is obtained fromblood of a pregnant female. Blood is collected using any standardtechnique for blood-drawing including but not limited to venipuncture.For example, blood can be drawn from a vein from the inside of the elbowor the back of the hand. Blood samples can be collected from a pregnantfemale at any time during fetal gestation. For example, blood samplescan be collected from human females at 1-4, 4-8, 8-12, 12-16, 16-20,20-24, 24-28, 28-32, 32-36, 36-40, or 40-44 weeks of fetal gestation,and preferably between 8-28 weeks of fetal gestation.

The blood sample is centrifuged to separate the plasma from the maternalcells. The plasma and maternal cell fractions are transferred toseparate tubes and re-centrifuged. The plasma fraction containscell-free fetal DNA and maternal DNA. Any standard DNA isolationtechnique can be used to isolate the fetal DNA and the maternal DNAincluding but not limited to QIAmp DNA Blood Midi Kit supplied by QIAGEN(Catalog number 51183).

In a preferred embodiment, blood can be collected into an apparatuscontaining a magnesium chelator including but not limited to EDTA, andis stored at 4° C. Optionally, a calcium chelator, including but notlimited to EGTA, can be added.

In another embodiment, a cell lysis inhibitor is added to the maternalblood including but not limited to formaldehyde, formaldehydederivatives, formalin, glutaraldehyde, glutaraldehyde derivatives, aprotein cross-linker, a nucleic acid cross-linker, a protein and nucleicacid cross-linker, primary amine reactive crosslinkers, sulfhydrylreactive crosslinkers, sulfhydryl addition or disulfide reduction,carbohydrate reactive crosslinkers, carboxyl reactive crosslinkers,photoreactive crosslinkers, cleavable crosslinkers, AEDP, APG, BASED,BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES, DFDNB, DMA, DMP,DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS,sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS, or compounds listed in Table XXIII.

In another embodiment, an agent that stabilizes cell membranes may beadded to the maternal blood samples to reduce maternal cell lysisincluding but not limited to aldehydes, urea formaldehyde, phenolformaldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterolderivatives, high concentrations of magnesium, vitamin E, and vitamin Ederivatives, calcium, calcium gluconate, taurine, niacin, hydroxylaminederivatives, bimoclomol, sucrose, astaxanthin, glucose, amitriptyline,isomer A hopane tetral phenylacetate, isomer B hopane tetralphenylacetate, citicoline, inositol, vitamin B, vitamin B complex,cholesterol hemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone,vitamin K, vitamin K complex, menaquinone, zonegran, zinc, ginkgo bilobaextract, diphenylhydantoin, perftoran, polyvinylpyrrolidone,phosphatidylserine, tegretol, PABA, disodium cromglycate, nedocromilsodium, phenyloin, zinc citrate, mexitil, dilantin, sodium hyaluronate,or polaxamer 188.

In another embodiment, the template DNA is obtained from the plasma orserum of the blood of the pregnant female. The percentage of fetal DNAin maternal plasma is between 0.39-11.9% (Pertl, and Bianchi, Obstetricsand Gynecology 98: 483-490 (2001)). The majority of the DNA in theplasma sample is maternal, which makes using the DNA for genotyping thefetus difficult. However, methods that increase the percentage of fetalDNA in the maternal plasma allow the sequence of the fetal DNA to bedetermined, and allow for the detection of genetic disorders includingmutations, insertions, deletions, and chromosomal abnormalities. Theaddition of cell lysis inhibitors, cell membrane stabilizers orcross-linkers to the maternal blood sample can increase the relativepercentage of fetal DNA. While lysis of both maternal and fetal cells isinhibited, the vast majority of cells are maternal, and thus by reducingthe lysis of maternal cells, there is a relative increase in thepercentage of free fetal DNA. See Example 4.

In another embodiment, any blood drawing technique, method, protocol, orequipment that reduce the amount of cell lysis can be used, includingbut not limited to a large boar needle, a shorter length needle, aneedle coating that increases laminar flow, e.g., teflon, a modificationof the bevel of the needle to increase laminar flow, or techniques thatreduce the rate of blood flow. The fetal cells likely are destroyed inthe maternal blood by the mother's immune system. However, it is likelythat a large portion of the maternal cell lysis occurs as a result ofthe blood draw or processing of the blood sample. Thus, methods thatprevent or reduce cell lysis will reduce the amount of maternal DNA inthe sample, and increase the relative percentage of free fetal DNA.

In another embodiment, an agent that preserves or stabilizes thestructural integrity of cells can be used to reduce the amount of celllysis.

In another embodiment, any protocol that reduces the amount of freematernal DNA in the maternal blood can be used prior to obtaining thesample. In another embodiment, prior to obtaining the sample, thepregnant female rests without physical activity for a period of timeincluding but not limited to 0-5, 5-10, 10-15, 15-20, 20-25, 25-30,30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-120, 120-180, 180-240,240-300, 300-360, 360-420, 420-480, 480-540, 540-600, 600-660, 660-720,720-780, 780-840, 840-900, 900-1200, 1200-1500, 1500-1800, 1800-2100,2100-2400, 2400-2700, 2700-3000, 3000-3300, 3300-3600, 3600-3900,3900-4200, 4200-4500, and greater than 4500 minutes. In anotherembodiment, the sample is obtained from the pregnant female after herbody has reached a relaxed state. The period of rest prior to obtainingthe sample may reduce the amount of maternal nucleic acid in the sample.In another embodiment, the sample is obtained from the pregnant femalein the a.m., including but not limited to 4-5 am, 5-6 am, 6-7 am, 7-8am, 8-9 am, 9-10 am, 10-11 am, and 11-12 am.

In another embodiment, the sample is obtained from the pregnant femaleafter she has slept for a period of time including but not limited to0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, orgreater than 12 hours.

In another embodiment, prior to obtaining the sample, the pregnantfemale exercises for a period of time followed by a period of rest. Inanother embodiment, the period of exercise includes but is not limitedto 0-15, 15-30, 30-45, 45-60, 60-120, 120-240, or greater than 240minutes.

In another embodiment, agents that prevent the destruction of DNA,including but not limited to a DNase inhibitor, zinc chloride,ethylenediaminetetraacetic acid, guanidine-HCl, guanidineisothiocyanate, N-lauroylsarcosine, and Na-dodecylsulphate, can be addedto the blood sample.

In another embodiment, fetal DNA is obtained from a fetal cell, whereinsaid fetal cell can be isolated from sources including but not limitedto maternal blood, umbilical cord blood, chorionic amniotic fluid,embryonic tissues and mucous obtained from the cervix or vagina of themother.

In a preferred embodiment, fetal cells are isolated from maternalperipheral blood. An antibody specific for fetal cells can be used topurify the fetal cells from the maternal serum (Mueller et al., Lancet336: 197-200 (1990); Ganshirt-Ahlert et al., Am. J. Obstet. Gynecol.166: 1350-1355 (1992)). Flow cytometry techniques can also be used toenrich fetal cells (Herzenberg et al., PNAS 76: 1453-1455 (1979);Bianchi et al., PNAS 87: 3279-3283 (1990); Bruch et al., PrenatalDiagnosis 11: 787-798 (1991)). U.S. Pat. No. 5,432,054 also describes atechnique for separation of fetal nucleated red blood cells, using atube having a wide top and a narrow, capillary bottom made ofpolyethylene. Centrifugation using a variable speed program results in astacking of red blood cells in the capillary based on the density of themolecules. The density fraction containing low density red blood cells,including fetal red blood cells, is recovered and then differentiallyhemolyzed to preferentially destroy maternal red blood cells. A densitygradient in a hypertonic medium is used to separate red blood cells, nowenriched in the fetal red blood cells from lymphocytes and rupturedmaternal cells. The use of a hypertonic solution shrinks the red bloodcells, which increases their density, and facilitate purification fromthe more dense lymphocytes. After the fetal cells have been isolated,fetal DNA can be purified using standard techniques in the art.

The nucleic acid that is to be analyzed can be any nucleic acid, e.g.,genomic, plasmid, cosmid, yeast artificial chromosomes, artificial orman-made DNA, including unique DNA sequences, and also DNA that has beenreverse transcribed from an RNA sample, such as cDNA. The sequence ofRNA can be determined according to the invention if it is capable ofbeing made into a double stranded DNA form to be used as template DNA.

The terms “primer” and “oligonucleotide primer” are interchangeable whenused to discuss an oligonucleotide that anneals to a template and can beused to prime the synthesis of a copy of that template.

“Amplified” DNA is DNA that has been “copied” once or multiple times,e.g. by polymerase chain reaction. When a large amount of DNA isavailable to assay, such that a sufficient number of copies of the locusof interest are already present in the sample to be assayed, it may notbe necessary to “amplify” the DNA of the locus of interest into an evenlarger number of replicate copies. Rather, simply “copying” the templateDNA once using a set of appropriate primers, which may contain hairpinstructures that allow the restriction enzyme recognition sites to bedouble stranded, can suffice.

“Copy” as in “copied DNA” refers to DNA that has been copied once, orDNA that has been amplified into more than one copy.

In one embodiment, the nucleic acid is amplified directly in theoriginal sample containing the source of nucleic acid. It is notessential that the nucleic acid be extracted, purified or isolated; itonly needs to be provided in a form that is capable of being amplified.Hybridization of the nucleic acid template with primer, prior toamplification, is not required. For example, amplification can beperformed in a cell or sample lysate using standard protocols well knownin the art. DNA that is on a solid support, in a fixed biologicalpreparation, or otherwise in a composition that contains non-DNAsubstances and that can be amplified without first being extracted fromthe solid support or fixed preparation or non-DNA substances in thecomposition can be used directly, without further purification, as longas the DNA can anneal with appropriate primers, and be copied,especially amplified, and the copied or amplified products can berecovered and utilized as described herein.

In a preferred embodiment, the nucleic acid is extracted, purified orisolated from non-nucleic acid materials that are in the original sampleusing methods known in the art prior to amplification.

In another embodiment, the nucleic acid is extracted, purified orisolated from the original sample containing the source of nucleic acidand prior to amplification, the nucleic acid is fragmented using anynumber of methods well known in the art including but not limited toenzymatic digestion, manual shearing, or sonication. For example, theDNA can be digested with one or more restriction enzymes that have arecognition site, and especially an eight base or six base pairrecognition site, which is not present in the loci of interest.Typically, DNA can be fragmented to any desired length, including 50,100, 250, 500, 1,000, 5,000, 10,000, 50,000 and 100,000 base pairs long.In another embodiment, the DNA is fragmented to an average length ofabout 1000 to 2000 base pairs. However, it is not necessary that the DNAbe fragmented.

Fragments of DNA that contain the loci of interest can be purified fromthe fragmented DNA before amplification. Such fragments can be purifiedby using primers that will be used in the amplification (see “PrimerDesign” section below) as hooks to retrieve the loci of interest, basedon the ability of such primers to anneal to the loci of interest. In apreferred embodiment, tag-modified primers are used, such as e.g.biotinylated primers.

By purifying the DNA fragments containing the loci of interest, thespecificity of the amplification reaction can be improved. This willminimize amplification of nonspecific regions of the template DNA.Purification of the DNA fragments can also allow multiplex PCR(Polymerase Chain Reaction) or amplification of multiple loci ofinterest with improved specificity.

The loci of interest that are to be sequenced can be selected based uponsequence alone. In humans, over 1.42 million single nucleotidepolymorphisms (SNPs) have been described (Nature 409:928-933 (2001); TheSNP Consortium LTD). On the average, there is one SNP every 1.9 kb ofhuman genome. However, the distance between loci of interest need not beconsidered when selecting the loci of interest to be sequenced accordingto the invention. If more than one locus of interest on genomic DNA isbeing analyzed, the selected loci of interest can be on the samechromosome or on different chromosomes.

In a preferred embodiment, the selected loci of interest can beclustered to a particular region on a chromosome. Multiple loci ofinterest can be located within a region of DNA such that even with anybreakage or fragmentation of the DNA, the multiple loci of interestremain linked. For example, if the DNA is obtained and by natural forcesis broken into fragments of 5 Kb, multiple loci of interest can beselected within the 5 Kb regions. This allows each fragment, as measuredby the loci of interest within that fragment, to serve as anexperimental unit, and will reduce any possible experimental noise ofcomparing loci of interest on multiple chromosomes.

The loci of interest on a chromosome can be any distance from each otherincluding but not limited to 10-50, 50-100, 100-150, 150-200, 200-250,250-500, 500-750, 750-1000, 1060-1500, 1500-2000, 2000-2500, 2500-3000,3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-10,000 and greater than10,000 base pairs.

In a preferred embodiment, the length of sequence that is amplified ispreferably different for each locus of interest so that the loci ofinterest can be separated by size.

In fact, it is an advantage of the invention that primers that copy anentire gene sequence need not be utilized. Rather, the copied locus ofinterest is preferably only a small part of the total gene or a smallpart of a non-coding region of DNA. There is no advantage to sequencingthe entire gene as this can increase cost and delay results. Sequencingonly the desired bases or loci of interest maximizes the overallefficiency of the method because it allows for the sequence of themaximum number of loci of interest to be determined in the fastestamount of time and with minimal cost.

Because a large number of sequences can be analyzed together, the methodof the invention is especially amenable to the large-scale screening ofa number of loci of interest.

Any number of loci of interest can be analyzed and processed, especiallyat the same time, using the method of the invention. The sample(s) canbe analyzed to determine the sequence at one locus of interest or atmultiple loci of interest at the same time. The loci of interest can bepresent on a single chromosome or on multiple chromosomes.

Alternatively, 2, 3, 4, 5, 6, 7, 8, 9, 10-20, 20-25, 25-30, 30-35,35-40, 40-45, 45-50, 50-100, 100-250, 250-500, 500-1,000, 1,000-2,000,2,000-3,000, 3,000-5,000, 5,000-10,000, 10,000-50,000 or more than50,000 loci of interest can be analyzed at the same time when a globalgenetic screening is desired. Such a global genetic screening might bedesired when using the method of the invention to provide a geneticfingerprint to identify an individual or for SNP genotyping.

The locus of interest to be copied can be within a coding sequence oroutside of a coding sequence. Preferably, one or more loci of interestthat are to be copied are within a gene. In a preferred embodiment, thetemplate DNA that is copied is a locus or loci of interest that iswithin a genomic coding sequence, either intron or exon. In a highlypreferred embodiment, exon DNA sequences are copied. The loci ofinterest can be sites where mutations are known to cause disease orpredispose to a disease state. The loci of interest can be sites ofsingle nucleotide polymorphisms. Alternatively, the loci of interestthat are to be copied can be outside of the coding sequence, forexample, in a transcriptional regulatory region, and especially apromoter, enhancer, or repressor sequence.

Method for Determining the Sequence of a Locus of Interest

Any method that provides information on the sequence of a nucleic acidcan be used including but not limited to allele specific PCR, PCR, gelelectrophoresis, ELISA, mass spectrometry, MALDI-TOF mass spectrometryhybridization, primer extension, fluorescence detection, fluorescenceresonance energy transfer (FRET), fluorescence polarization, DNAsequencing, Sanger dideoxy sequencing, DNA sequencing gels, capillaryelectrophoresis on an automated DNA sequencing machine, microchannelelectrophoresis, microarray, southern blot, slot blot, dot blot, singleprimer linear nucleic acid amplification, as described in U.S. Pat. No.6,251,639, SNP-IT, GeneChips, HuSNP, BeadArray, TaqMan assay, Invaderassay, MassExtend, or MassCleave™ (hMC) method.

The preferred method of determining the sequence has previously beendescribed in U.S. application Ser. No. 10/093,618, filed on Mar. 11,2002, hereby incorporated by reference in its entirety.

I. Primer Design

Published sequences, including consensus sequences, can be used todesign or select primers for use in amplification of template DNA. Theselection of sequences to be used for the construction of primers thatflank a locus of interest can be made by examination of the sequence ofthe loci of interest, or immediately thereto. The recently publishedsequence of the human genome provides a source of useful consensussequence information from which to design primers to flank a desiredhuman gene locus of interest.

By “flanking” a locus of interest is meant that the sequences of theprimers are such that at least a portion of the 3′ region of one primeris complementary to the antisense strand of the template DNA andupstream from the locus of interest site (forward primer), and at leasta portion of the 3′ region of the other primer is complementary to thesense strand of the template DNA and downstream of the locus of interest(reverse primer). A “primer pair” is intended a pair of forward andreverse primers. Both primers of a primer pair anneal in a manner thatallows extension of the primers, such that the extension results inamplifying the template DNA in the region of the locus of interest.

Primers can be prepared by a variety of methods including but notlimited to cloning of appropriate sequences and direct chemicalsynthesis using methods well known in the art (Narang et al. MethodsEnzymol. 68:90 (1979); Brown et al., Methods Enzymol. 68:109 (1979)).Primers can also be obtained from commercial sources such as OperonTechnologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies.The primers can have an identical melting temperature. The lengths ofthe primers can be extended or shortened at the 5′ end or the 3′ end toproduce primers with desired melting temperatures. In a preferredembodiment, one of the primers of the prime pair is longer than theother primer. In a preferred embodiment, the 3′ annealing lengths of theprimers, within a primer pair, differ. Also, the annealing position ofeach primer pair can be designed such that the sequence and length ofthe primer pairs yield the desired melting temperature. The simplestequation for determining the melting temperature of primers smaller than25 base pairs is the Wallace Rule (Td=2(A+T)+4(G+C)). Computer programscan also be used to design primers, including but not limited to ArrayDesigner Software (Arrayit Inc.), Oligonucleotide Probe Sequence DesignSoftware for Genetic Analysis (Olympus Optical Co.), NetPrimer, andDNAsis from Hitachi Software Engineering. The TM (melting or annealingtemperature) of each primer is calculated using software programs suchas Net Primer (free web based program athttp://premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html;Internet address as of Apr. 17, 2002).

In another embodiment, the annealing temperature of the primers can berecalculated and increased after any cycle of amplification, includingbut not limited to cycle 1, 2, 3, 4, 5, cycles 6-10, cycles 10-15,cycles 15-20, cycles 20-25, cycles 25-30, cycles 30-35, or cycles 35-40.After the initial cycles of amplification, the 5′ half of the primers isincorporated into the products from each loci of interest, thus the TMcan be recalculated based on both the sequences of the 5′ half and the3′ half of each primer.

For example, in FIG. 1B, the first cycle of amplification is performedat about the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the second primer (region “c”), which is 13 bases.After the first cycle, the annealing temperature can be raised to TM2,which is about the melting temperature of the 3′ region, which annealsto the template DNA, of the first primer, which is depicted as region“b.” The second primer cannot bind to the original template DNA becauseit only anneals to 13 bases in the original DNA template, and TM2 isabout the melting temperature of approximately 20 bases, which is the 3′annealing region of the first primer (FIG. 1C). However, the firstprimer can bind to the DNA that was copied in the first cycle of thereaction. In the third cycle, the annealing temperature is raised toTM3, which is about the melting temperature of the entire sequence ofthe second primer, which is depicted as regions “c” and “d.” The DNAtemplate produced from the second cycle of PCR contains both regions c′and d′, and therefore, the second primer can anneal and extend at TM3(FIG. 1D). The remaining cycles are performed at TM3. The entiresequence of the first primer (a+b′) can anneal to the template from thethird cycle of PCR, and extend (FIG. 1E). Increasing the annealingtemperature will decrease non-specific binding and increase thespecificity of the reaction, which is especially useful if amplifying alocus of interest from human genomic DNA, which is about 3×10⁹ basepairs long.

As used herein, the term “about” with regard to annealing temperaturesis used to encompass temperatures within 10 degrees celsius of thestated temperatures.

In one embodiment, one primer pair is used for each locus of interest.However, multiple primer pairs can be used for each locus of interest.

In one embodiment, primers are designed such that one or both primers ofthe primer pair contain sequence in the 5′ region for one or morerestriction endonucleases (restriction enzyme).

As used herein, with regard to the position at which restriction enzymesdigest DNA, the “sense” strand is the strand reading 5′ to 3′ in thedirection in which the restriction enzyme cuts. For example, BsmF Irecognizes the following sequences:

5′ GGGAC(N)₁₀ 3′ (SEQ ID NO: 1) 5′ (N)₁₄GTCCC 3′ (SEQ ID NO: 2)3′ CCCTG(N)₁₄ 5′ (SEQ ID NO: 2) 3′(N)₁₀CAGGG 5′ (SEQ ID NO: 1)

The sense strand is the strand containing the “GGGAC” sequence as itreads 5′ to 3′ in the direction that the restriction enzyme cuts.

As used herein, with regard to the position at which restriction enzymesdigest DNA, the “antisense” strand is the strand reading 3′ to 5′ in thedirection in which the restriction enzyme cuts.

In another embodiment, one of the primers in a primer pair is designedsuch that it contains a restriction enzyme recognition site for arestriction enzyme that cuts “n” nucleotides away from the recognitionsite, and produces a recessed 3′ end and a 5′ overhang that contains thelocus of interest (herein referred to as a “second primer”). “N” is adistance from the recognition site to the site of the cut by therestriction enzyme. In other words, the second primer of a primer paircontains a recognition site for a restriction enzyme that does not cutDNA at the recognition site but cuts “n” nucleotides away from therecognition site. For example, if the recognition sequence is for therestriction enzyme BceA I, the enzyme will cut ten (10) nucleotides fromthe recognition site on the sense strand, and twelve (12) nucleotidesaway from the recognition site on the antisense strand.

The 3′ region and preferably, the 3′ half, of the primers is designed toanneal to a sequence that flanks the loci of interest (FIG. 1A). Thesecond primer can anneal any distance from the locus of interestprovided that digestion with the restriction enzyme that recognizes therestriction enzyme recognition site on this primer generates a 5′overhang that contains the locus of interest. The 5′ overhangs can be ofany size, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, and morethan 8 bases.

In a preferred embodiment, the 3′ end of the primer that anneals closerto the locus of interest (second primer) can anneal 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or more than 14 bases from the locus ofinterest or at the locus of interest.

In a preferred embodiment, the second primer is designed to annealcloser to the locus of interest than the other primer of a primer pair(the other primer is herein referred to as a “first primer”). The secondprimer can be a forward or reverse primer and the first primer can be areverse or forward primer, respectively. Whether the first or secondprimer should be the forward or reverse primer can be determined bywhich design will provide better sequencing results.

For example, the primer that anneals closer to the locus of interest cancontain a recognition site for the restriction enzyme BsmF I, which cutsten (10) nucleotides from the recognition site on the sense strand, andfourteen (14) nucleotides from the recognition site on the antisensestrand. In this case, the primer can be designed so that the restrictionenzyme recognition site is 13 bases, 12 bases, 10 bases or 11 bases fromthe locus of interest. If the recognition site is 13 bases from thelocus of interest, digestion with BsmF I will generate a 5′ overhang(RXXX), wherein the locus of interest (R) is the first nucleotide in theoverhang (reading 3′ to 5′), and X is any nucleotide. If the recognitionsite is 12 bases from the locus of interest, digestion with BsmF I willgenerate a 5′ overhang (XRXX), wherein the locus of interest (R) is thesecond nucleotide in the overhang (reading 3′ to 5′). If the recognitionsite is 11 bases from the locus of interest, digestion with BsmF I willgenerate a 5′ overhang (XXRX), wherein the locus of interest (R) is thethird nucleotide in the overhang (reading 3′ to 5′). The distancebetween the restriction enzyme recognition site and the locus ofinterest should be designed so that digestion with the restrictionenzyme generates a 5′ overhang, which contains the locus of interest.The effective distance between the recognition site and the locus ofinterest will vary depending on the choice of restriction enzyme.

In another embodiment, the primer that anneals closer to the locus ofinterest site, relative to the other primer, can be designed so that therestriction enzyme that generates the 5′ overhang, which contains thelocus of interest, will see the same sequence at the cut site,independent of the nucleotide at the locus of interest site. Forexample, if the primer that anneals closer to the locus of interest isdesigned so that the recognition site for the restriction enzyme BsmF I(5′ GGGAC 3′) is thirteen bases from the locus of interest, therestriction enzyme will cut the antisense strand one base from the locusof interest. The nucleotide at the locus of interest is adjacent to thecut site, and may vary from DNA molecule to DNA molecule. If it isdesired that the nucleotides adjacent to the cut site be identical, theprimer can be designed so that the restriction enzyme recognition sitefor BsmF I is twelve bases away from the locus of interest site.Digestion with BsmF I will generate a 5′ overhang, wherein the locus ofinterest site is in the second position of the overhang (reading 3′ to5′) and is no longer adjacent to the cut site. Designing the primer sothat the restriction enzyme recognition site is twelve (12) bases fromthe locus of interest site allows the nucleotides adjacent to the cutsite to be the same, independent of the nucleotide at the locus ofinterest. Also, primers that have been designed so that the restrictionenzyme recognition site, BsmF I, is eleven (11) or ten (10) bases fromthe locus of interest site will allow the nucleotides adjacent to thecut site to be the same, independent of the nucleotide at the locus ofinterest. Similar strategies of primer design can be employed with otherrestriction enzymes so that the nucleotides adjacent to the cut sitewill be the same, independent of the nucleotide at the loci of interest.

The 3′ end of the first primer (either the forward or the reverse) canbe designed to anneal at a chosen distance from the locus of interest.Preferably, for example, this distance is between 1-10, 10-25, 25-50,50-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400,400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800,800-850, 850-900, 900-950, 950-1000 and greater than 1000 bases awayfrom the locus of interest. The annealing sites of the first primers arechosen such that each successive upstream primer is further and furtheraway from its respective downstream primer.

For example, if at locus of interest 1 the 3′ ends of the first andsecond primers are Z bases apart, then at locus of interest 2, the 3′ends of the upstream and downstream primers are Z+K bases apart, whereK=1, 2, 3, 4, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80,80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,700-800, 800-900, 900-1000, or greater than 1000 bases (FIG. 2). Thepurpose of making the first primers further and further apart from theirrespective second primers is so that the PCR products of all the loci ofinterest differ in size and can be separated, e.g., on a sequencing gel.This allows for multiplexing by pooling the PCR products in later steps.

In one embodiment, the 5′ region of the first or second primer can havea recognition site for any type of restriction enzyme. In a preferredembodiment, the 5′ region of the first and/or second primer has at leastone restriction enzyme recognition site that is different from therestriction enzyme recognition site that is used to generate the 5′overhang, which contains the locus of interest.

In one embodiment, the 5′ region of the first primer can have arecognition site for any type of restriction enzyme. In a preferredembodiment, the first primer has at least one restriction enzymerecognition site that is different from the restriction enzymerecognition site in the second primer. In another preferred embodiment,the first primer anneals further away from the locus of interest thanthe second primer.

In a preferred embodiment, the second primer contains a restrictionenzyme recognition sequence for a Type IIS restriction enzyme includingbut not limited to BceA I and BsmF I, which produce a two base 5′overhang and a four base 5′ overhang, respectively. Restriction enzymesthat are Type IIS are preferred because they recognize asymmetric basesequences (not palindromic like the orthodox Type II enzymes). Type IISrestriction enzymes cleave DNA at a specified position that is outsideof the recognition site, typically up to 20 base pairs outside of therecognition site. These properties make Type IIS restriction enzymes,and the recognition sites thereof, especially useful in the method ofthe invention. Preferably, the Type IIS restriction enzymes used in thismethod leave a 5′ overhang and a recessed 3′.

A wide variety of Type IIS restriction enzymes are known and suchenzymes have been isolated from bacteria, phage, archeabacteria andviruses of eukaryotic algae and are commercially available (Promega,Madison Wis.; New England Biolabs, Beverly, Mass.; Szybalski W. et al.,Gene 100:13-26, 1991). Examples of Type IIS restriction enzymes thatwould be useful in the method of the invention include, but are notlimited to enzymes such as those listed in Table I.

TABLE I Recognition/ Enzyme- Source Cleavage Site Supplier Alw I -Acinetobacter lwoffii GGATC(4/5) NE Bioiabs Alw26 I - Acinetobacterlwojfi GTCTC(1/5) Promega Bbs I - Bacillus laterosporus GAAGAC(2/6) NEBioiabs Bbv I - Bacillus brevis GCAGC(8/12) NE Bioiabs BceA I - Bacilluscereus 1315 IACGGC(12/14) NE Bioiabs Bmr I - Bacillus megaleriumCTGGG(5/4) NE Bioiabs Bsa I - Bacillus stearothermophilus 6-55GGTCTC(1/5) NE Bioiabs Bst71 I - Bacillus stearothermophilus 71GCAGC(8/12) Promega BsmA I - Bacillus stearothermophilus A664 GTCTC(1/5)NE Bioiabs BsmB I -Bacillus stearothermophilus B61 CGTCTC(1/5) NEBioiabs BsmF I - Bacillus stearothermophilus F GGGAC(10/14) NE BioiabsBspM I - Bacillus species M ACCTGC(4/8) NE Bioiabs Ear I - Enterobacteraerogenes CTCTTC(1/4) NE Biolabs Fau I - Flavobacterium aquatileCCCGC(4/6) NE Biolabs Fok I - Flavobacterium okeonokoites GGATG(9/13) NEBiolabs Hga I - Haemophilus gallinarum GACGC(5/10) NE Biolabs Ple I -Pseudomonas lemoignei GAGTC(4/5) NE Biolabs Sap I - Saccharopolysporaspecies GCTCTTC(1/4) NE Biolabs SfaN I - Streptococcus faecalis ND547GCATC(5/9) NE Biolabs Sth132 I - Streptococcus thermophilus STI32CCCG(4/8) No commercial supplier (Gene 195: 201-206 (1997))

In one embodiment, a primer pair has sequence at the 5′ region of eachof the primers that provides a restriction enzyme recognition site thatis unique for one restriction enzyme.

In another embodiment, a primer pair has sequence at the 5′ region ofeach of the primers that provide a restriction site that is recognizedby more than one restriction enzyme, and especially for more than oneType IIS restriction enzyme. For example, certain consensus sequencescan be recognized by more than one enzyme. For example, BsgI, Eco571 andBpmI all recognize the consensus (G/C)TGnAG and cleave 16 by away on theantisense strand and 14 by away on the sense strand. A primer thatprovides such a consensus sequence would result in a product that has asite that can be recognized by any of the restriction enzymes BsgI,Eco571 and BpmI.

Other restriction enzymes that cut DNA at a distance from therecognition site, and produce a recessed 3′ end and a 5′ overhanginclude Type III restriction enzymes.

For example, the restriction enzyme EcoP15I recognizes the sequence 5′CAGCAG 3′ and cleaves 25 bases downstream on the sense strand and 27bases on the antisense strand. It will be further appreciated by aperson of ordinary skill in the art that new restriction enzymes arecontinually being discovered and can readily be adopted for use in thesubject invention.

In another embodiment, the second primer can contain a portion of therecognition sequence for a restriction enzyme, wherein the fullrecognition site for the restriction enzyme is generated uponamplification of the template DNA such that digestion with therestriction enzyme generates a 5′ overhang containing the locus ofinterest. For example, the recognition site for BsmF I is 5′ GGGACN₁₀^(↓) 3′ (SEQ ID NO: 1). The 3′ region, which anneals to the templateDNA, of the second primer can end with the nucleotides “GGG,” which donot have to be complementary with the template DNA. If the 3′ annealingregion is about 10-20 bases, even if the last three bases do not anneal,the primer will extend and, generate a BsmF I site.

Second primer: (SEQ ID NO: 3) 5′ GGAAATTCCATGATGCGTGGG→ Template DNA(SEQ ID NO: 27) 3′ CCTTTAAGGTACTACGCAN₁N₂N₃TG 5′ (SEQ ID NO: 4)5′ GGAAATTCCATGATGCCTN₁,N₂,N₃,AC 3′

The second primer can be designed to anneal to the template DNA, whereinthe next two bases of the template DNA are thymidine and guanine, suchthat an adenosine and cytosine are incorporated into the primer forminga recognition site for BsmF I, 5′ GGGACN₁₀ ^(↓) 3′ (SEQ ID NO: 1). Thesecond primer can be designed to anneal in such a manner that digestionwith BsmF I generates a 5′ overhang containing the locus of interest.

In another embodiment, the second primer can contain an entire or fullrecognition site for a restriction enzyme or a portion of a recognitionsite, which generates a full recognition site upon primer-dependentreplication of the template DNA such that digestion with a restrictionenzyme that cuts at the recognition site and generates a 5′ overhangthat contains the locus of interest. For example, the restriction enzymeBsaJ I binds the following recognition site: 5′ C^(↓)CN₁N₂GG 3′. Thesecond primer can be designed such that the 3′ region, which anneals tothe template DNA of the primer ends with “CC”, the SNP of interest isrepresented by “N₁”, and the template sequence downstream of the SNP is“N₂GG.”

Second primer: (SEQ ID NO: 5) 5′ GGAAATTCCATGATGCGTACC→ Template DNA(SEQ ID NO: 28) 3′ CCTTTAAGGTACTACGCATGGN₁N₂CC 5′ (SEQ ID NO: 6)5′ GGAAATTCCATGATGCCTACCN₁,N₂,GG 3′

After digestion with BsaJ I, a 5′ overhang of the following sequencewould be generated:

5′ C 3′ 3′ GGN₁N₂CC 5′

If the nucleotide guanine is not reported at the locus of interest, the3′ recessed end can be filled in with unlabeled cytosine, which iscomplementary to the first nucleotide in the overhang. After removingthe excess cytosine, labeled ddNTPs can be used to fill in the nextnucleotide, N₁, which represents the locus of interest. Otherrestriction enzymes can be used including but not limited to BssK I (5′^(↓)CCNGG 3′), Dde I (5′ C^(↓)TNAG 3′), EcoN I (5′ CCTNN^(↓)NNNAGG 3′(SEQ ID NO: 7)), Fnu4H I (5′ GC^(↓)NGC 3′), Hinf I (5′ G^(↓)ANTC 3′)PflF I (5′ GACN^(↓)NNGTC 3′), Sau96 I (5′ G^(↓)GNCC 3′), ScrF I (5′CC^(↓)NGG 3′), and Tth1 11 I (5′ GACN^(↓)NNGTC 3′).

It is not necessary that the 3′ region, which anneals to the templateDNA, of the second primer be 100% complementary to the template DNA. Forexample, the last 1, 2, or 3 nucleotides of the 3′ end of the secondprimer can be mismatches with the template DNA. The region of the primerthat anneals to the template DNA will target the primer, and allow theprimer to extend. Even if the last two nucleotides are not complementaryto the template DNA, the primer will extend and generate a restrictionenzyme recognition site. For example, the last two nucleotides in thesecond primer are “CC.” The second primer anneals to the template DNA,and allows extension even if “CC” is not complementary to thenucleotides Na, and Nb, on the template DNA.

Second primer: (SEQ ID NO: 5) 5′ GGAAATTCCATGATGCGTACC→ Template DNA(SEQ ID NO: 29) 3′ CCTTTAAGGTACTACGCATN_(a),N_(b),N₁,N₂,CC 5′ (SEQ IDNO: 8) 5′ GGAAATTCCATGATGCCTAN_(a)N_(b)N₁N₂GG 3′

After digestion with BsaJ I, a 5′ overhang of the following sequencewould be generated:

5′ C 3′ 3′ GGN₁N₂CC 5′

If the nucleotide guanine is not reported at the locus of interest, the5′ overhang can be filled in with unlabeled cytosine. The excesscytosine can be rinsed away, and filled in with labeled ddNTPs. Thefirst nucleotide incorporated (N₁′) corresponds to the locus ofinterest. If guanine is reported at the locus of interest, the loci ofinterest can be filled in with unlabeled cytosine and a nucleotidedownstream of the locus of interest can be detected. For example, assumeN₂ is adenine. If the locus of interest is guanine, unlabeled cytosinecan be used in the fill in reaction. After removing the cytosine, a fillin reaction with labeled thymidine can be used. The labeled thymidinewill be incorporated only if the locus of interest was a guanine. Thus,the sequence of the locus of interest can be determined by detecting anucleotide downstream of the locus of interest.

In another embodiment, the first and second primers contain a portion ofa recognition sequence for a restriction enzyme, wherein the fullrecognition site for the restriction enzyme is generated uponamplification of the template DNA such that digestion with therestriction enzyme generates a 5′ overhang containing the locus ofinterest. The recognition site for any restriction enzyme that containsone or more than one variable nucleotide can be generated including butnot limited to the restriction enzymes BssK I (5′^(↓)CCNGG 3′), Dde I(5′C^(↓)TNAG 3′), Econ I (5′CCTNN^(↓)NNNAGG 3′ (SEQ ID NO: 7)), Fnu4H I(5′GC^(↓)NGC 3′), Hinf I (5′G^(↓)ANTC 3′), PflF I (5′ GACN^(↓)NNGTC 3′),Sau96 I (5′ G^(↓)GNCC 3′), ScrF I (5′ CC^(↓)NGG 3′), and Tth1 11 I (5′GACN^(↓)NNGTC 3′).

In a preferred embodiment, the 3′ regions of the first and secondprimers contain the partial sequence for a restriction enzyme, whereinthe partial sequence contains 1, 2, 3, 4 or more than 4 mismatches withthe template DNA; these mismatches create the restriction enzymerecognition site. The number of mismatches that can be tolerated at the3′ end depends on the length of the primer. For example, if the locus ofinterest is represented by N₁, a first primer can be designed to becomplementary to the template DNA, depicted below as region “a.” The 3′region of the first primer ends with “CC,” which is not complementary tothe template DNA. The second primer is designed to be complementary tothe template DNA, which is depicted below as region “b′”. The 3′ regionof the second primer ends with “CC,” which is not complementary to thetemplate DNA.

After one round of amplification the following products would begenerated:

In cycle two, the primers can anneal to the templates that weregenerated from the first cycle of PCR:

After cycle two of PCR, the following products would be generated:

The restriction enzyme recognition site for BsaJ I is generated, andafter digestion with BsaJ 1, a 5′ overhang containing the locus ofinterest is created. The locus of interest can be detected as describedin detail below.

In another embodiment, a primer pair has sequence at the 5′ region ofeach of the primers that provides two or more restriction sites that arerecognized by two or more restriction enzymes.

In a most preferred embodiment, a primer pair has different restrictionenzyme recognition sites at the 5′ regions, especially 5′ ends, suchthat a different restriction enzyme is required to cleave away anyundesired sequences. For example, the first primer for locus of interest“A” can contain sequence recognized by a restriction enzyme, “X,” whichcan be any type of restriction enzyme, and the second primer for locusof interest “A,” which anneals closer to the locus of interest, cancontain sequence for a restriction enzyme, “Y,” which is a Type IISrestriction enzyme that cuts “n” nucleotides away and leaves a 5′overhang and a recessed 3′ end. The 5′ overhang contains the locus ofinterest. After binding the amplified DNA to streptavidin coated wells,one can digest with enzyme “Y,” rinse, then fill in with labelednucleotides and rinse, and then digest with restriction enzyme “X,”which will release the DNA fragment containing the locus of interestfrom the solid matrix. The locus of interest can be analyzed bydetecting the labeled nucleotide that was “filled in” at the locus ofinterest, e.g. SNP site.

In another embodiment, the second primers for the different loci ofinterest that are being amplified according to the invention containrecognition sequence in the 5′ regions for the same restriction enzymeand likewise all the first primers also contain the same restrictionenzyme recognition site, which is a different enzyme from the enzymethat recognizes the second primers.

In another embodiment, the second primers for the multiple loci ofinterest that are being amplified according to the invention containrestriction enzyme recognition sequences in the 5′ regions for differentrestriction enzymes.

In another embodiment, the first primers for the multiple loci ofinterest that are being amplified according to the invention containrestriction enzyme recognition sequences in the 5′ regions for differentrestriction enzymes. Multiple restriction enzyme sequences provide anopportunity to influence the order in which pooled loci of interest arereleased from the solid support. For example, if 50 loci of interest areamplified, the first primers can have a tag at the extreme 5′ end to aidin purification and a restriction enzyme recognition site, and thesecond primers can contain a recognition site for a type IIS restrictionenzyme. For example, several of the first primers can have a restrictionenzyme recognition site for EcoR I, other first primers can have arecognition site for Pst I, and still other first primers can have arecognition site for BamH I. After amplification, the loci of interestcan be bound to a solid support with the aid of the tag on the firstprimers. By performing the restriction digests one restriction enzyme ata time, one can serially release the amplified loci of interest. If thefirst digest is performed with EcoR I, the loci of interest amplifiedwith the first primers containing the recognition site for EcoR I willbe released, and collected while the other loci of interest remain boundto the solid support. The amplified loci of interest can be selectivelyreleased from the solid support by digesting with one restriction enzymeat a time. The use of different restriction enzyme recognition sites inthe first primers allows a larger number of loci of interest to beamplified in a single reaction tube.

In a preferred embodiment, any region 5′ of the restriction enzymedigestion site of each primer can be modified with a functional groupthat provides for fragment manipulation, processing, identification,and/or purification. Examples of such functional groups, or tags,include but are not limited to biotin, derivatives of biotin,carbohydrates, haptens, dyes, radioactive molecules, antibodies, andfragments of antibodies, peptides, and immunogenic molecules.

In another embodiment, the template DNA can be replicated once, withoutbeing amplified beyond a single round of replication. This is usefulwhen there is a large amount of the DNA available for analysis such thata large number of copies of the loci of interest are already present inthe sample, and further copies are not needed. In this embodiment, theprimers are preferably designed to contain a “hairpin” structure in the5′ region, such that the sequence doubles back and anneals to a sequenceinternal to itself in a complementary manner. When the template DNA isreplicated only once, the DNA sequence comprising the recognition sitewould be single-stranded if not for the “hairpin” structure. However, inthe presence of the hairpin structure, that region is effectively doublestranded, thus providing a double stranded substrate for activity byrestriction enzymes.

To the extent that the reaction conditions are compatible, all theprimer pairs to analyze a locus or loci of interest of DNA can be mixedtogether for use in the method of the invention. In a preferredembodiment, all primer pairs are mixed with the template DNA in a singlereaction vessel. Such a reaction vessel can be, for example, a reactiontube, or a well of a microtiter plate.

Alternatively, to avoid competition for nucleotides and to minimizeprimer dimers and difficulties with annealing temperatures for primers,each locus of interest or small groups of loci of interest can beamplified in separate reaction tubes or wells, and the products laterpooled if desired. For example, the separate reactions can be pooledinto a single reaction vessel before digestion with the restrictionenzyme that generates a 5′ overhang, which contains the locus ofinterest or SNP site, and a 3′ recessed end. Preferably, the primers ofeach primer pair are provided in equimolar amounts. Also, especiallypreferably, each of the different primer pairs is provided in equimolaramounts relative to the other pairs that are being used.

In another embodiment, combinations of primer pairs that allow efficientamplification of their respective loci of interest can be used (see e.g.FIG. 2). Such combinations can be determined prior to use in the methodof the invention. Multi-well plates and PCR machines can be used toselect primer pairs that work efficiently with one another. For example,gradient PCR machines, such as the Eppendorf Mastercycler® gradient PCRmachine, can be used to select the optimal annealing temperature foreach primer pair. Primer pairs that have similar properties can be usedtogether in a single reaction tube.

In another embodiment, a multi-sample container including but notlimited to a 96-well or more plate can be used to amplify a single locusof interest with the same primer pairs from multiple template DNAsamples with optimal PCR conditions for that locus of interest.Alternatively, a separate multi-sample container can be used foramplification of each locus of interest and the products for eachtemplate DNA sample later pooled. For example, gene A from 96 differentDNA samples can be amplified in microtiter plate 1, gene B from 96different DNA samples can be amplified in microtiter plate 2, etc., andthen the amplification products can be pooled.

The result of amplifying multiple loci of interest is a preparation thatcontains representative PCR products having the sequence of each locusof interest. For example, if DNA from only one individual is used as thetemplate DNA and if hundreds of disease-related loci of interest wereamplified from the template DNA, the amplified DNA would be a mixture ofsmall, PCR products from each of the loci of interest. Such apreparation could be further analyzed at that time to determine thesequence at each locus of interest or at only some loci of interest.Additionally, the preparation could be stored in a manner that preservesthe DNA and can be analyzed at a later time. Information contained inthe amplified DNA can be revealed by any suitable method including butnot limited to fluorescence detection, sequencing, gel electrophoresis,and mass spectrometry (see “Detection of Incorporated Nucleotide”section below).

II. Amplification of Loci of Interest

The template DNA can be amplified using any suitable method known in theart including but not limited to PCR (polymerase chain reaction), 3SR(self-sustained sequence reaction), LCR (ligase chain reaction),RACE-PCR (rapid amplification of cDNA ends), PLCR (a combination ofpolymerase chain reaction and ligase chain reaction), Q-beta phageamplification (Shah et al., J. Medical Micro. 33: 1435-41 (1995)), SDA(strand displacement amplification), SOE-PCR (splice overlap extensionPCR), and the like. These methods can be used to design variations ofthe releasable primer mediated cyclic amplification reaction explicitlydescribed in this application. In the most preferred embodiment, thetemplate DNA is amplified using PCR (PCR: A Practical Approach, M. J.McPherson, et al., IRL Press (1991); PCR Protocols: A Guide to Methodsand Applications, Innis, et al., Academic Press (1990); and PCRTechnology: Principals and Applications of DNA Amplification, H. A.Erlich, Stockton Press (1989)). PCR is also described in numerous U.S.patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792, 5,023,171;5,091,310; and 5,066,584.

The components of a typical PCR reaction include but are not limited toa template DNA, primers, a reaction buffer (dependent on choice ofpolymerase), dNTPs (dATP, dTTP, dGTP, and dCTP) and a DNA polymerase.Suitable PCR primers can be designed and prepared as discussed above(see “Primer Design” section above). Briefly, the reaction is heated to95° C. for 2 min. to separate the strands of the template DNA, thereaction is cooled to an appropriate temperature (determined bycalculating the annealing temperature of designed primers) to allowprimers to anneal to the template DNA, and heated to 72° C. for twominutes to allow extension.

In a preferred embodiment, the annealing temperature is increased ineach of the first three cycles of amplification to reduce non-specificamplification. See also Example 1, below. The TM1 of the first cycle ofPCR is about the melting temperature of the 3′ region of the secondprimer that anneals to the template DNA. The annealing temperature canbe raised in cycles 2-10, preferably in cycle 2, to TM2, which is aboutthe melting temperature of the 3′ region, which anneals to the templateDNA, of the first primer. If the annealing temperature is raised incycle 2, the annealing temperature remains about the same until the nextincrease in annealing temperature. Finally, in any cycle subsequent tothe cycle in which the annealing temperature was increased to TM2,preferably cycle 3, the annealing temperature is raised to TM3, which isabout the melting temperature of the entire second primer. After thethird cycle, the annealing temperature for the remaining cycles can beat about TM3 or can be further increased. In this example, the annealingtemperature is increased in cycles 2 and 3. However, the annealingtemperature can be increased from a low annealing temperature in cycle 1to a high annealing temperature in cycle 2 without any further increasesin temperature or the annealing temperature can progressively changefrom a low annealing temperature to a high annealing temperature in anynumber of incremental steps. For example, the annealing temperature canbe changed in cycles 2, 3, 4, 5, 6, etc.

After annealing, the temperature in each cycle is increased to an“extension” temperature to allow the primers to “extend” and thenfollowing extension the temperature in each cycle is increased to thedenaturization temperature. For PCR products less than 500 base pairs insize, one can eliminate the extension step in each cycle and just havedenaturization and annealing steps. A typical PCR reaction consists of25-45 cycles of denaturation, annealing and extension as describedabove. However, as previously noted, one cycle of amplification (onecopy) can be sufficient for practicing the invention.

In another embodiment, multiple sets of primers wherein a primer setcomprises a forward primer and a reverser primer, can be used to amplifythe template DNA for 1-5, 5-10, 10-15, 15-20 or more than 20 cycles, andthen the amplified product is further amplified in a reaction with asingle primer set or a subset of the multiple primer sets. In apreferred embodiment, a low concentration of each primer set is used tominimize primer-dimer formation. A low concentration of starting DNA canbe amplified using multiple primer sets. Any number of primer sets canbe used in the first amplification reaction including but not limitingto 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100,100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-1000, and greater than 1000. In another embodiment, the amplifiedproduct is amplified in a second reaction with a single primer set. Inanother embodiment, the amplified product is further amplified with asubset of the multiple primer pairs including but not limited to 2-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150,150-200, 200-250, and more than 250.

The multiple primer sets will amplify the loci of interest, such that aminimal amount of template DNA is not limiting for the number of locithat can be detected. For example, if template DNA is isolated from asingle cell or the template DNA is obtained from a pregnant female,which comprises both maternal template DNA and fetal template DNA, lowconcentrations of each primer set can be used in a first amplificationreaction to amplify the loci of interest. The low concentration ofprimers reduces the formation of primer-dimer and increases theprobability that the primers will anneal to the template DNA and allowthe polymerase to extend. The optimal number of cycles performed withthe multiple primer sets is determined by the concentration of theprimers. Following the first amplification reaction, additional primerscan be added to further amplify the loci of interest. Additional amountsof each primer set can be added and further amplified in a singlereaction. Alternatively, the amplified product can be further amplifiedusing a single primer set in each reaction or a subset of the multipleprimers sets. For example, if 150 primer sets were used in the firstamplification reaction, subsets of 10 primer sets can be used to furtheramplify the product from the first reaction.

Any DNA polymerase that catalyzes primer extension can be used includingbut not limited to E. coli DNA polymerase, Klenow fragment of E. coliDNA polymerase 1, T7 DNA polymerase, T4 DNA polymerase, Taq polymerase,Pfu DNA polymerase, Vent. DNA polymerase, bacteriophage 29, REDTaq™Genomic DNA polymerase, or sequenase. Preferably, a thermostable DNApolymerase is used. A “hot start” PCR can also be performed wherein thereaction is heated to 95° C. for two minutes prior to addition of thepolymerase or the polymerase can be kept inactive until the firstheating step in cycle 1. “Hot start” PCR can be used to minimizenonspecific amplification. Any number of PCR cycles can be used toamplify the DNA, including but not limited to 2, 5, 10, 15, 20, 25, 30,35, 40, or 45 cycles. In a most preferred embodiment, the number of PCRcycles performed is such that equimolar amounts of each loci of interestare produced.

III. Purification of Amplified DNA

Purification of the amplified DNA is not necessary for practicing theinvention. However, in one embodiment, if purification is preferred, the5′ end of the primer (first or second primer) can be modified with a tagthat facilitates purification of the PCR products. In a preferredembodiment, the first primer is modified with a tag that facilitatespurification of the PCR products. The modification is preferably thesame for all primers, although different modifications can be used if itis desired to separate the PCR products into different groups.

The tag can be any chemical moiety including but not limited to aradioisotope, fluorescent reporter molecule, chemiluminescent reportermolecule, antibody, antibody fragment, hapten, biotin, derivative ofbiotin, photobiotin, iminobiotin, digoxigenin, avidin, enzyme,acridinium, sugar, enzyme, apoenzyme, homopolymeric oligonucleotide,hormone, ferromagnetic moiety, paramagnetic moiety, diamagnetic moiety,phosphorescent moiety, luminescent moiety, electrochemiluminescentmoiety, chromatic moiety, moiety having a detectable electron spinresonance, electrical capacitance, dielectric constant or electricalconductivity, or combinations thereof.

As one example, the 5′ ends of the primers can be biotinylated (Kandpalet al., Nucleic Acids Res. 18:1789-1795 (1990); Kaneoka et al.,Biotechniques 10:30-34 (1991); Green et al., Nucleic Acids Res.18:6163-6164 (1990)). The biotin provides an affinity tag that can beused to purify the copied DNA from the genomic DNA or any other DNAmolecules that are not of interest. Biotinylated molecules can bepurified using a streptavidin coated matrix as shown in FIG. 1F,including but not limited to Streptawell, transparent, High-Bind platesfrom Roche Molecular Biochemicals (catalog number 1 645 692, as listedin Roche Molecular Biochemicals, 2001 Biochemicals Catalog).

The PCR product of each locus of interest is placed into separate wellsof a Streptavidin coated plate. Alternatively, the PCR products of theloci of interest can be pooled and placed into a streptavidin coatedmatrix, including but not limited to the Streptawell, transparent,High-Bind plates from Roche Molecular Biochemicals (catalog number 1 645692, as listed in Roche Molecular Biochemicals, 2001 BiochemicalsCatalog).

The amplified DNA can also be separated from the template DNA usingnon-affinity methods known in the art, for example, by polyacrylamidegel electrophoresis using standard protocols.

IV. Digestion of Amplified DNA

The amplified DNA can be digested with a restriction enzyme thatrecognizes a sequence that had been provided on the first or secondprimer using standard protocols known within the art (FIGS. 6A-6D).Restriction enzyme digestions are performed using standard protocolswell known within the art. The enzyme used depends on the restrictionrecognition site generated with the first or second primer. See “PrimerDesign” section, above, for details on restriction recognition sitesgenerated on primers.

Type IIS restriction enzymes are extremely useful in that they cutapproximately 10-20 base pairs outside of the recognition site.Preferably, the Type IIS restriction enzymes used are those thatgenerate a 5′ overhang and a recessed 3′ end, including but not limitedto BceA I and BsmF I (see e.g. Table I). In a most preferred embodiment,the second primer (either forward or reverse) contains a restrictionenzyme recognition sequence for BsmF I or BceA I. The Type IISrestriction enzyme BsmF I recognizes the nucleic acid sequence GGGAC,and cuts 14 nucleotides from the recognition site on the antisensestrand and 10 nucleotides from the recognition site on the sense strand.Digestion with BsmF I generates a 5′ overhang of four (4) bases.

For example, if the second primer is designed so that afteramplification the restriction enzyme recognition site is 13 bases fromthe locus of interest, then after digestion, the locus of interest isthe first base in the 5′ overhang (reading 3′ to 5′), and the recessed3′ end is one base from the locus of interest. The 3′ recessed end canbe filled in with a nucleotide that is complementary to the locus ofinterest. One base of the overhang can be filled in usingdideoxynucleotides. However, 1, 2, 3, or 4 bases of the overhang can befilled in using deoxynucleotides or a mixture of dideoxynucleotides anddeoxynucleotides.

The restriction enzyme BsmF I cuts DNA ten (10) nucleotides from therecognition site on the sense strand and fourteen (14) nucleotides fromthe recognition site on the antisense strand. However, in a sequencedependent manner, the restriction enzyme BsmF I also cuts eleven (11)nucleotides from the recognition site on the sense strand and fifteen(15) nucleotides from the recognition site on the antisense strand.Thus, two populations of DNA molecules exist after digestion: DNAmolecules cut at 10/14 and DNA molecules cut at 11/15. If therecognition site for BsmF I is 13 bases from the locus of interest inthe amplified product, then DNA molecules cut at the 11/15 position willgenerate a 5′ overhang that contains the locus of interest in the secondposition of the overhang (reading 3′ to 5′). The 3′ recessed end of theDNA molecules can be filled in with labeled nucleotides. For example, iflabeled dideoxynucleotides are used, the 3′ recessed end of themolecules cut at 11/15 would be filled in with one base, whichcorresponds to the base upstream from the locus of interest, and the 3′recessed end of molecules cut at 10/14 would be filled in with one base,which corresponds to the locus of interest. The DNA molecules that havebeen cut at the 10/14 position and the DNA molecules that have been cutat the 11/15 position can be separated by size, and the incorporatednucleotides detected. This allows detection of both the nucleotidebefore the locus of interest, detection of the locus of interest, andpotentially the three bases after the locus of interest.

Alternatively, if the base upstream from the locus of interest and thelocus of interest are different nucleotides, then the 3′ recessed end ofthe molecules cut at 11/15 can be filled in with deoxynucleotide that iscomplementary to the upstream base. The remaining deoxynucleotide iswashed away, and the locus of interest site can be filled in with eitherlabeled deoxynucleotides, unlabeled deoxynucleotides, labeleddideoxynucleotides, or unlabeled dideoxynucleotides. After the fill inreaction, the nucleotide can be detected by any suitable method. Thus,after the first fill in reaction with dNTP, the 3′ recessed end of themolecules cut at 10/14 and 11/15 is upstream from the locus of interest.The 3′ recessed end can now be filled in one base, which corresponds tothe locus of interest, two bases, three bases or four bases.

The restriction enzyme BceA I recognizes the nucleic acid sequence ACGGCand cuts 12 (twelve) nucleotides from the recognition site on the sensestrand and 14 (fourteen) nucleotides from the recognition site on theantisense strand. If the distance from the recognition site for BceA Ion the second primer is designed to be thirteen (13) bases from thelocus of interest (see FIGS. 4A-4D), digestion with BceA I will generatea 5′ overhang of two bases, which contains the locus of interest, and arecessed 3′ end that is upstream from the locus of interest. The locusof interest is the first nucleotide in the 5′ overhang (reading 3′ to5′).

Alternative cutting is also seen with the restriction enzyme BceA I,although at a much lower frequency than is seen with BsmF I. Therestriction enzyme BceA I can cut thirteen (13) nucleotides from therecognition site on the sense strand and fifteen (15) nucleotides fromthe recognition site on the antisense strand. Thus, two populations ofDNA molecules exist: DNA molecules cut at 12/14 and DNA molecules cut at13/15. If the restriction enzyme recognition site is 13 bases from thelocus of interest in the amplified product, DNA molecules cut at the13/15 position yield a 5′ overhang, which contains the locus of interestin the second position of the overhang (reading 3′ to 5′). Labeleddideoxynucleotides can be used to fill in the 3′ recessed end of the DNAmolecules. The DNA molecules cut at 13/15 will have the base upstreamfrom the locus of interest filled in, and the DNA molecules cut at 12/14will have the locus of interest site filled in. The DNA molecules cut at13/15 and those cut at 12/14 can be separated by size, and theincorporated nucleotide detected. Thus, the alternative cutting can beused to obtain additional sequence information.

Alternatively, if the two bases in the 5′ overhang are different, the 3′recessed end of the DNA molecules, which were cut at 13/15, can befilled in with the deoxynucleotide complementary to the first base inthe overhang, and excess deoxynucleotide washed away. After filling in,the 3′ recessed end of the DNA molecules that were cut at 12/14 and theDNA molecules that were cut at 13/15 are upstream from the locus ofinterest. The 3′ recessed ends can be filled with either labeleddideoxynucleotides, unlabeled dideoxynucleotides, labeleddeoxynucleotides, or unlabeled deoxynucleotides.

If the primers provide different restriction sites for certain of theloci of interest that were copied, all the necessary restriction enzymescan be added together to digest the copied DNA simultaneously.Alternatively, the different restriction digests can be made insequence, for example, using one restriction enzyme at a time, so thatonly the product that is specific for that restriction enzyme isdigested.

Optimal restriction enzyme digestion conditions, including but notlimited to the concentration of enzyme, temperature, buffer conditions,and the time of digestion can be optimized for each restriction enzyme.For example, the alternative cutting seen with the type IIS restrictionenzyme BsmF I can be reduced, if desired, by performing the restrictionenzyme digestion at lower temperatures including but not limited to25-16°, 16-12° C., 12-8° C., 8-4° C., or 4-0° C.

V. Incorporation of Labeled Nucleotides

Digestion with the restriction enzyme that recognizes the sequence onthe second primer generates a recessed 3′ end and a 5′ overhang, whichcontains the locus of interest (FIG. 1G). The recessed 3′ end can befilled in using the 5′ overhang as a template in the presence ofunlabeled or labeled nucleotides or a combination of both unlabeled andlabeled nucleotides. The nucleotides can be labeled with any type ofchemical group or moiety that allows for detection including but notlimited to radioactive molecules, fluorescent molecules, antibodies,antibody fragments, haptens, carbohydrates, biotin, derivatives ofbiotin, phosphorescent moieties, luminescent moieties,electrochemiluminescent moieties, chromatic moieties, and moietieshaving a detectable electron spin resonance, electrical capacitance,dielectric constant or electrical conductivity. The nucleotides can belabeled with one or more than one type of chemical group or moiety. Eachnucleotide can be labeled with the same chemical group or moiety.Alternatively, each different nucleotide can be labeled with a differentchemical group or moiety. The labeled nucleotides can be dNTPs, ddNTPs,or a mixture of both dNTPs and ddNTPs. The unlabeled nucleotides can bedNTPs, ddNTPs or a mixture of both dNTPs and ddNTPs.

Any combination of nucleotides can be used to incorporate nucleotidesincluding but not limited to unlabeled deoxynucleotides, labeleddeoxynucleotides, unlabeled dideoxynucleotides, labeleddideoxynucleotides, a mixture of labeled and unlabeled deoxynucleotides,a mixture of labeled and unlabeled dideoxynucleotides, a mixture oflabeled deoxynucleotides and labeled dideoxynucleotides, a mixture oflabeled deoxynucleotides and unlabeled dideoxynucleotides, a mixture ofunlabeled deoxynucleotides and unlabeled dideoxynucleotides, a mixtureof unlabeled deoxynucleotides and labeled dideoxynucleotides,dideoxynucleotide analogues, deoxynucleotide analogues, a mixture ofdideoxynucleotide analogues and deoxynucleotide analogues,phosphorylated nucleoside analogues, 2′-deoxynucleotide-5′-triphosphate,and modified 2′-deoxynucleotide-5′-triphosphate.

For example, as shown in FIG. 1H, in the presence of a polymerase, the3′ recessed end can be filled in with fluorescent ddNTP using the 5′overhang as a template. The incorporated ddNTP can be detected using anysuitable method including but not limited to fluorescence detection.

All four nucleotides can be labeled with different fluorescent groups,which will allow one reaction to be performed in the presence of allfour labeled nucleotides. Alternatively, four separate “fill in”reactions can be performed for each locus of interest; each of the fourreactions will contain a different labeled nucleotide (e.g. ddATP*,ddTTP*, ddGTP*, or ddCTP*, where * indicates a labeled nucleotide). Eachnucleotide can be labeled with different chemical groups or the samechemical groups. The labeled nucleotides can be dideoxynucleotides ordeoxynucleotides.

In another embodiment, nucleotides can be labeled with fluorescent dyesincluding but not limited to fluorescein, pyrene, 7-methoxycoumarin,Cascade Blue™, Alexa Flur 350, Alexa Flur 430, Alexa Flur 488, AlexaFlur 532, Alexa Flur 546, Alexa Flur 568, Alexa Flur 594, Alexa Flur633, Alexa Flur 647, Alexa Flur 660, Alexa Flur 680, AMCA-X,dialkylaminocoumarin, Pacific Blue, Marina Blue, BODIPY 493/503, BODIPYF1-X, DTAF, Oregon Green 500, Dansyl-X, 6-FAM, Oregon Green 488, OregonGreen 514, Rhodamine Green-X, Rhodol Green, Calcein, Eosin, ethidiumbromide, NBD, TET, 2′,4′,5′,7′ tetrabromosulfonefluorescien, BODIPY R6G,BODIPY-F1 BR2, BODIPY 530/550, HEX, BODIPY 558/568, BODIPY-TMR-X.,PyMPO, BODIPY 564/570, TAMRA, BODIPY 576/589, Cy3, Rhodamine Red-x,BODIPY 581/591, carboxyXrhodamine, Texas Red-X, BODIPY-TR-X., Cy5,SpectrumAqua, SpectrumGreen #1, SpectrumGreen #2, SpectrumOrange,SpectrumRed, or naphthofluorescein.

In another embodiment, the “fill in” reaction can be performed withfluorescently labeled dNTPs, wherein the nucleotides are labeled withdifferent fluorescent groups. The incorporated nucleotides can bedetected by any suitable method including but not limited toFluorescence Resonance Energy Transfer (FRET).

In another embodiment, a mixture of both labeled ddNTPs and unlabeleddNTPs can be used for filling in the recessed 3′ end of the SNP or locusof interest. Preferably, the 5′ overhang consists of more than one base,including but not limited to 2, 3, 4, 5, 6 or more than 6 bases. Forexample, if the 5′ overhang consists of the sequence “XGAA,” wherein Xis the locus of interest, e.g. SNP, then filling in with a mixture oflabeled ddNTPs and unlabeled dNTPs will produce several different DNAfragments. If a labeled ddNTP is incorporated at position “X,” thereaction will terminate and a single labeled base will be incorporated.If however, an unlabeled dNTP is incorporated, the polymerase continuesto incorporate other bases until a labeled ddNTP is incorporated. If thefirst two nucleotides incorporated are dNTPs, and the third is a ddNTP,the 3′ recessed end will be extend by three bases. This DNA fragment canbe separated from the other DNA fragments that were extended by 1, 2, or4 bases by size. A mixture of labeled ddNTPs and unlabeled dNTPs willallow all bases of the overhang to be filled in, and provides additionalsequence information about the locus of interest, e.g. SNP (see FIGS. 7Eand 9D).

After incorporation of the labeled nucleotide, the amplified DNA can bedigested with a restriction enzyme that recognizes the sequence providedby the first primer. For example, in FIG. 1I, the amplified DNA isdigested with a restriction enzyme that binds to region “a,” whichreleases the DNA fragment containing the incorporated nucleotide fromthe streptavidin matrix.

Alternatively, one primer of each primer pair for each locus of interestcan be attached to a solid support matrix including but not limited to awell of a microliter plate. For example, streptavidin-coated microtiterplates can be used for the amplification reaction with a primer pair,wherein one primer is biotinylated. First, biotinylated primers arebound to the streptavidin-coated microtiter plates. Then, the plates areused as the reaction vessel for PCR amplification of the loci ofinterest. After the amplification reaction is complete, the excessprimers, salts, and template DNA can be removed by washing. Theamplified DNA remains attached to the microtiter plate. The amplifiedDNA can be digested with a restriction enzyme that recognizes a sequenceon the second primer and generates a 5′ overhang, which contains thelocus of interest. The digested fragments can be removed by washing.After digestion, the SNP site or locus of interest is exposed in the 5′overhang. The recessed 3′ end is filled in with a labeled nucleotide,including but not limited to, fluorescent ddNTP in the presence of apolymerase. The labeled DNA can be released into the supernatant in themicrotiter plate by digesting with a restriction enzyme that recognizesa sequence in the 5′ region of the first primer.

In another embodiment, one nucleotide can be used to determine thesequence of multiple alleles of a gene. A nucleotide that terminates theelongation reaction can be used to determine the sequence of multiplealleles of a gene. At one allele, the terminating nucleotide iscomplementary to the locus of interest in the 5′ overhang of saidallele. The nucleotide is incorporated and terminates the reaction. At adifferent allele, the terminating nucleotide is not complementary to thelocus of interest, which allows a non-terminating nucleotide to beincorporated at the locus of interest of the different allele. However,the terminating nucleotide is complementary to a nucleotide downstreamfrom the locus of interest in the 5′ overhang of said different allele.The sequence of the alleles can be determined by analyzing the patternsof incorporation of the terminating nucleotide. The terminatingnucleotide can be labeled or unlabeled.

In a another embodiment, the terminating nucleotide is a nucleotide thatterminates or hinders the elongation reaction including but not limitedto a dideoxynucleotide, a dideoxynucleotide derivative, adideoxynucleotide analog, a dideoxynucleotide homolog, adideoxynucleotide with a sulfur chemical group, a deoxynucleotide, adeoxynucleotide derivative, a deoxynucleotide homolog, a deoxynucleotideanalog, a deoxynucleotide with a sulfur chemical group, arabinosidetriphosphate, an arabinoside triphosphate analog, an arabinosidetriphosphate homolog, or an arabinoside derivative.

In another embodiment, a terminating nucleotide labeled with one signalgenerating moiety tag, including but not limited to a fluorescent dye,can be used to determine the sequence of the alleles of a locus ofinterest. The use of a single nucleotide labeled with one signalgenerating moiety tag eliminates any difficulties that can arise whenusing different fluorescent moieties. In addition, using one nucleotidelabeled with one signal generating moiety tag to determine the sequenceof alleles of a locus of interest reduces the number of reactions, andeliminates pipetting errors.

For example, if the second primer contains the restriction enzymerecognition site for BsmFI, digestion will generate a 5′ overhang of 4bases. The second primer can be designed such that the locus of interestis located in the first position of the overhang. A representativeoverhang is depicted below, where R represents the locus of interest:

5′ CAC 3′ GTG R T G G Overhang position 1 2 3 4

One nucleotide with one signal generating moiety tag can be used todetermine whether the variable site is homozygous or heterozygous. Forexample, if the variable site is adenine (A) or guanine (G), then eitheradenine or guanine can be used to determine the sequence of the allelesof the locus of interest, provided that there is an adenine or guaninein the overhang at position 2, 3, or 4.

For example, if the nucleotide in position 2 of the overhang isthymidine, which is complementary to adenine, then labeled ddATP,unlabeled dCTP, dGTP, and dTTP can be used to determine the sequence ofthe alleles of the locus of interest. The ddATP can be labeled with anysignal generating moiety including but not limited to a fluorescent dye.If the template DNA is homozygous for adenine, then labeled ddATP* willbe incorporated at position 1 complementary to the overhang at thealleles, and no nucleotide incorporation will be seen at position 2, 3or 4 complementary to the overhang.

Allele 1 5′ CCC A* 3′ GGG T T G G Overhang position 1 2 3 4 Allele 25′ CCC A* 3′ GGG T T G G Overhang position 1 2 3 4

One signal will be seen corresponding to incorporation of labeled ddATPat position 1 complementary to the overhang, which indicates that theindividual is homozygous for adenine at this position. This method oflabeling eliminates any difficulties that may arise from using differentdyes that have different quantum coefficients.

Homozygous Guanine:

If the template DNA is homozygous for guanine, then no ddATP will beincorporated at position 1 complementary to the overhang, but ddATP willbe incorporated at the first available position, which in this case isposition 2 complementary to the overhang. For example, if the secondposition in the overhang corresponds to a thymidine, then:

Allele 1 5′ CCC G A* 3′ GGG C T G G Overhang position 1 2 3 4 Allele 25′ CCC G A* 3′ GGG C T G G Overhang position 1 2 3 4

One signal will be seen corresponding to incorporation of ddATP atposition 2 complementary to the overhang, which indicates that theindividual is homozygous for guanine. The molecules that are filled inat position 2 complementary to the overhang will have a differentmolecular weight than the molecules filled in at position 1complementary to the overhang.

Heterozygous Condition;

Allele 1 5′ CCC A* 3′ GGG T T G G Overhang position 1 2 3 4 Allele 25′ CCC G A* 3′ GGG C T G G Overhang position 1 2 3 4

Two signals will be seen; the first signal corresponds to the ddATPfilled in at position one complementary to the overhang and the secondsignal corresponds to the ddATP filled in at position 2 complementary tothe overhang. The two signals can be separated based on molecularweight; allele 1 and allele 2 will be separated by a single base pair,which allows easy detection and quantitation of the signals. Moleculesfilled in at position one can be distinguished from molecules filled inat position two using any method that discriminates based on molecularweight including but not limited to gel electrophoresis, capillary gelelectrophoresis, DNA sequencing, and mass spectrometry. It is notnecessary that the nucleotide be labeled with a chemical moiety; the DNAmolecules corresponding to the different alleles can be separated basedon molecular weight.

If position 2 of the overhang is not complementary to adenine, it ispossible that positions 3 or 4 may be complementary to adenine. Forexample, position 3 of the overhang may be complementary to thenucleotide adenine, in which case labeled ddATP may be used to determinethe sequence of both alleles.

Homozygous for Adenine:

Allele 1 5′ CCC A* 3′ GGG T G T G Overhang position 1 2 3 4 Allele 25′ CCC A* 3′ GGG T G T G Overhang position 1 2 3 4

Homozygous for Guanine:

Allele 1 5′ CCC G C A* 3′ GGG C G T G Overhang position 1 2 3 4 Allele 25′ CCC G C A* 3′ GGG C G T G Overhang position 1 2 3 4

Heterozygous:

Allele 1 5′ CCC A* 3′ GGG T G T G Overhang position 1 2 3 4 Allele 25′ CCC G C A* 3′ GGG C G T G Overhang position 1 2 3 4

Two signals will be seen; the first signal corresponds to the ddATPfilled in at position 1 complementary to the overhang and the secondsignal corresponds to the ddATP filled in at position 3 complementary tothe overhang. The two signals can be separated based on molecularweight; allele 1 and allele 2 will be separated by two bases, which canbe detected using any method that discriminates based on molecularweight.

Alternatively, if positions 2 and 3 are not complementary to adenine(i.e positions 2 and 3 of the overhang correspond to guanine, cytosine,or adenine) but position 4 is complementary to adenine, labeled ddATPcan be used to determine the sequence of both alleles.

Homozygous for Adenine:

Allele 1 5′ CCC A* 3′ GGG T G G T Overhang position 1 2 3 4 Allele 25′ CCC A* 3′ GGG T G G T Overhang position 1 2 3 4

One signal will be seen that corresponds to the molecular weight ofmolecules filled in with ddATP at position one complementary to theoverhang, which indicates that the individual is homozygous for adenineat the variable site.

Homozygous for Guanine:

Allele 1 5′ CCC G C C A* 3′ GGG C G G T Overhang position 1 2 3 4 Allele2 5′ CCC G C C A* 3′ GGG C G G T Overhang position 1 2 3 4

One signal will be seen that corresponds to the molecular weight ofmolecules filled in at position 4 complementary to the overhang, whichindicates that the individual is homozygous for guanine.

Heterozygous:

Allele 1 5′ CCC A* 3′ GGG T G G T Overhang position 1 2 3 4 Allele 25′ CCC G C C A* 3′ GGG C G G T Overhang position 1 2 3 4

Two signals will be seen; the first signal corresponds to the ddATPfilled in at position one complementary to the overhang and the secondsignal corresponds to the ddATP filled in at position 4 complementary tothe overhang. The two signals can be separated based on molecularweight; allele 1 and allele 2 will be separated by three bases, whichallows detection and quantitation of the signals. The molecules filledin at position 1 and those filled in at position 4 can be distinguishedbased on molecular weight.

As discussed above, if the variable site contains either adenine orguanine, either labeled adenine or labeled guanine can be used todetermine the sequence of both alleles. If positions 2, 3, or 4 of theoverhang are not complementary to adenine but one of the positions iscomplementary to a guanine, then labeled ddGTP can be used to determinewhether the template DNA is homozygous or heterozygous for adenine orguanine. For example, if position 3 in the overhang corresponds to acytosine then the following signals will be expected if the template DNAis homozygous for guanine, homozygous for adenine, or heterozygous:

Homozygous for Guanine:

Allele 1 5′ CCC G* 3′ GGG C T C T Overhang position 1 2 3 4 Allele 25′ CCC G* 3′ GGG C T C T Overhang position 1 2 3 4

One signal will be seen that corresponds to the molecular weight ofmolecules filled in with ddGTP at position one complementary to theoverhang, which indicates that the individual is homozygous for guanine.

Homozygous for Adenine:

Allele 1 5′ CCC A A G* 3′ GGG T T C T Overhang position 1 2 3 4 Allele 25′ CCC A A G* 3′ GGG T T C T Overhang position 1 2 3 4

One signal will be seen that corresponds to the molecular weight ofmolecules filled in at position 3 complementary to the overhang, whichindicates that the individual is homozygous for adenine at the variablesite.

Heterozygous:

Allele 1 5′ CCC G* 3′ GGG C T C T Overhang position 1 2 3 4 Allele 25′ CCC A A G* 3′ GGG T T C T Overhang position 1 2 3 4

Two signals will be seen; the first signal corresponds to the ddGTPfilled in at position one complementary to the overhang and the secondsignal corresponds to the ddGTP filled in at position 3 complementary tothe overhang. The two signals can be separated based on molecularweight; allele 1 and allele 2 will be separated by two bases, whichallows easy detection and quantitation of the signals.

In another embodiment, the nucleotide labeled with a single chemicalmoiety, which is used to determine the sequence of alleles of interest,can be analyzed by a variety of methods including but not limited tofluorescence detection, DNA sequencing gel, capillary electrophoresis onan automated DNA sequencing machine, microchannel electrophoresis, andother methods of sequencing, mass spectrometry, time of flight massspectrometry, quadrupole mass spectrometry, magnetic sector massspectrometry, electric sector mass spectrometry infrared spectrometry,ultraviolet spectrometry, palentiostatic amperometry or by DNAhybridization techniques including Southern Blots, Slot Blots, DotBlots, and DNA microarrays, wherein DNA fragments would be useful asboth “probes” and “targets,” ELISA, fluorimetry, Fluorescence ResonanceEnergy Transfer (FRET), SNP-IT, GeneChips, HuSNP, BeadArray, TaqManassay, Invader assay, MassExtend, or MassCleave™ (hMC) method.

Some type IIS restriction enzymes also display alternative cutting asdiscussed above. For example, BsmFI will cut at 10/14 and 11/15 from therecognition site. However, the cutting patterns are not mutuallyexclusive; if the 11/15 cutting pattern is seen at a particularsequence, 10/14 cutting is also seen. If the restriction enzyme BsmF Icuts at 10/14 from the recognition site, the 5′ overhang will beX₁X₂X₃X₄. If BsmF I cuts 11/15 from the recognition site, the 5′overhang will be X₀X₁X₂X₃. If position X₀ of the overhang iscomplementary to the labeled nucleotide, the labeled nucleotide will beincorporated at position X₀ and provides an additional level of qualityassurance. It provides additional sequence information.

For example, if the variable site is adenine or guanine, and position 3in the overhang is complementary to adenine, labeled ddATP can be usedto determine the genotype at the variable site. If position 0 of the11/15 overhang contains the nucleotide complementary to adenine, ddATPwill be filled in and an additional signal will be seen.

Heterozygous:

10/14 Allele 1 5′ CCA A* 3′ GGT T G T G Overhang position 1 2 3 4 10/14Allele 2 5′ CCA G C A* 3′ GGT C G T G Overhang position 1 2 3 4 11/15Allele 1 5′ CC A* 3′ GG T T G T Overhang position 0 1 2 3 11/15 Allele 25′ CC A* 3′ GG T C G T Overhang position 0 1 2 3

Three signals are seen; one corresponding to the ddATP incorporated atposition 0 complementary to the overhang, one corresponding to the ddATPincorporated at position 1 complementary to the overhang, and onecorresponding to the ddATP incorporated at position 3 complementary tothe overhang. The molecules filled in at position 0, 1, and 3complementary to the overhang differ in molecular weight and can beseparated using any technique that discriminates based on molecularweight including but not limited to gel electrophoresis, and massspectrometry.

For quantitating the ratio of one allele to another allele or whendetermining the relative amount of a mutant DNA sequence in the presenceof wild type DNA sequence, an accurate and highly sensitive method ofdetection must be used. The alternate cutting displayed by type IISrestriction enzymes may increase the difficulty of determining ratios ofone allele to another allele because the restriction enzyme may notdisplay the alternate cutting (11/15) pattern on the two allelesequally. For example, allele 1 may be cut at 10/14 80% of the time, and11/15 20% of the time. However, because the two alleles may differ insequence, allele 2 may be cut at 10/14 90% of the time, and 11/15 20% ofthe time.

For purposes of quantitation, the alternate cutting problem can beeliminated when the nucleotide at position 0 of the overhang is notcomplementary to the labeled nucleotide. For example, if the variablesite corresponds to adenine or guanine, and position 3 of the overhangis complementary to adenine (i.e, a thymidine is located at position 3of the overhang), labeled ddATP can be used to determine the genotype ofthe variable site. If position 0 of the overhang generated by the 11/15cutting properties is not complementary to adenine, (i.e, position 0 ofthe overhang corresponds to guanine, cytosine, or adenine) no additionalsignal will be seen from the fragments that were cut 11/15 from therecognition site. Position 0 complementary to the overhang can be filledin with unlabeled nucleotide, eliminating any complexity seen from thealternate cutting pattern of restriction enzymes. This method provides ahighly accurate method for quantitating the ratio of a variable siteincluding but not limited to a mutation, or a single nucleotidepolymorphism.

For instance, if SNP X can be adenine or guanine, this method oflabeling allows quantitation of the alleles that correspond to adenineand the alleles that correspond to guanine, without determining if therestriction enzyme displays any differences between the alleles withregard to alternate cutting patterns.

Heterozygous:

10/14 Allele 1 5′ CCG A* 3′ GGC T G T G Overhang position 1 2 3 4 10/14Allele 2 5′ CCG G C A* 3′ GGC C G T G Overhang position 1 2 3 4

The overhang generated by the alternate cutting properties of BsmF I isdepicted below:

11/15 Allele 1 5′ CC 3′ GG C T G T Overhang position 0 1 2 3 11/15Allele 2 5′ CC 3′ GG C C G T Overhang position 0 1 2 3

After filling in with labeled ddATP and unlabeled dGTP, dCTP, dTTP, thefollowing molecules would be generated:

11/15 Allele 1 5′ CC G A* 3′ GG C T G T Overhang position 0 1 2 3 11/15Allele 2 5′ CC G G C A* 3′ GG C C G T Overhang position 0 1 2 3

Two signals are seen; one corresponding to the molecules filled in withddATP at position one complementary to the overhang and onecorresponding to the molecules filled in with ddATP at position 3complementary to the overhang. Position 0 of the 11/15 overhang isfilled in with unlabeled nucleotide, which eliminates any difficulty inquantitating a ratio for the nucleotide at the variable site on allele 1and the nucleotide at the variable site on allele 2.

Any nucleotide can be used including adenine, adenine derivatives,adenine homologues, guanine, guanine derivatives, guanine homologues,cytosine, cytosine derivatives, cytosine homologues, thymidine,thymidine derivatives, or thymidine homologues, or any combinations ofadenine, adenine derivatives, adenine homologues, guanine, guaninederivatives, guanine homologues, cytosine, cytosine derivatives,cytosine homologues, thymidine, thymidine derivatives, or thymidinehomologues.

The nucleotide can be labeled with any chemical group or moiety,including but not limited to radioactive molecules, fluorescentmolecules, antibodies, antibody fragments, haptens, carbohydrates,biotin, derivatives of biotin, phosphorescent moieties, luminescentmoieties, electrochemiluminescent moieties, chromatic moieties, andmoieties having a detectable electron spin resonance, electricalcapacitance, dielectric constant or electrical conductivity. Thenucleotide can be labeled with one or more than one type of chemicalgroup or moiety.

In another embodiment, labeled and unlabeled nucleotides can be used.Any combination of deoxynucleotides and dideoxynucleotides can be usedincluding but not limited to labeled dideoxynucleotides and labeleddeoxynucleotides; labeled dideoxynucleotides and unlabeleddeoxynucleotides; unlabeled dideoxynucleotides and unlabeleddeoxynucleotides; and unlabeled dideoxynucleotides and labeleddeoxynucleotides.

In another embodiment, nucleotides labeled with a chemical moiety can beused in the PCR reaction. Unlabeled nucleotides then are used to fill-inthe 5′ overhangs generated after digestion with the restriction enzyme.An unlabeled terminating nucleotide can be used to in the presence ofunlabeled nucleotides to determine the sequence of the alleles of alocus of interest.

For example, if labeled dTTP was used in the PCR reaction, the following5′ overhang would be generated after digestion with BsmF I:

10/14 Allele 1 5′ CT*G A 3′ GA C T G T G Overhang position 1 2 3 4 10/14Allele 2 5′ CT*G G C A 3′ GA C C G T G Overhang position 1 2 3 4

Unlabeled ddATP, unlabeled dCTP, unlabeled dGTP, and unlabeled dTTP canbe used to fill-in the 5′ overhang. Two signals will be generated; onesignal corresponds to the DNA molecules filled in with unlabeled ddATPat position 1 complementary to the overhang and the second signalcorresponds to DNA molecules filled in with unlabeled ddATP at position3 complementary to the overhang. The DNA molecules can be separatedbased on molecular weight and can be detected by the fluorescence of thedTTP, which was incorporated during the PCR reaction.

The labeled DNA loci of interest sites can be analyzed by a variety ofmethods including but not limited to fluorescence detection, DNAsequencing gel, capillary electrophoresis on an automated DNA sequencingmachine, microchannel electrophoresis, and other methods of sequencing,mass spectrometry, time of flight mass spectrometry, quadrupole massspectrometry, magnetic sector mass spectrometry, electric sector massspectrometry infrared spectrometry, ultraviolet spectrometry,palentiostatic amperometry or by DNA hybridization techniques includingSouthern Blots, Slot Blots, Dot Blots, and DNA microarrays, wherein DNAfragments would be useful as both “probes” and “targets,” ELISA,fluorimetry, Fluorescence Resonance Energy Transfer (FRET), SNP-IT,GeneChips, HuSNP, BeadArray, TaqMan assay, Invader assay, MassExtend, orMassCleave™ (hMC) method.

This method of labeling is extremely sensitive and allows the detectionof alleles of a locus of interest that are in various ratios includingbut not limited to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6-1:10, 1:11-1:20,1:21-1:30, 1:31-1:40, 1:41-1:50, 1:51-1:60, 1:61-1:70, 1:71-1:80,1:81-1:90, 1:91:1:100, 1:101-1:200, 1:250, 1:251-1:300, 1:301-1:400,1:401-1:500, 1:501-1:600, 1:601-1:700, 1:701-1:800, 1:801-1:900,1:901-1:1000, 1:1001-1:2000, 1:2001-1:3000, 1:3001-1:4000,1:4001-1:5000, 1:5001-1:6000, 1:6001-1:7000, 1:7001-1:8000,1:8001-1:9000, 1:9001-1:10,000; 1:10,001-1:20,000, 1:20,001:1:30,000,1:30,001-1:40,000, 1:40,001-1:50,000, and greater than 1:50,000.

For example, this method of labeling allows one nucleotide labeled withone signal generating moiety to be used to determine the sequence ofalleles at a SNP locus, or detect a mutant allele amongst a populationof normal alleles, or detect an allele encoding antibiotic resistancefrom a bacterial cell amongst alleles from antibiotic sensitivebacteria, or detect an allele from a drug resistant virus amongstalleles from drug-sensitive virus, or detect an allele from anon-pathogenic bacterial strain amongst alleles from a pathogenicbacterial strain.

As shown above, a single nucleotide can be used to determine thesequence of the alleles at a particular locus of interest. This methodis especially useful for determining if an individual is homozygous orheterozygous for a particular mutation or to determine the sequence ofthe alleles at a particular SNP site. This method of labeling eliminatesany errors caused by the quantum coefficients of various dyes. It alsoallows the reaction to proceed in a single reaction vessel including butnot limited to a well of a microtiter plate, or a single eppendorf tube.

This method of labeling is especially useful for the detection ofmultiple genetic signals in the same sample. For example, this method isuseful for the detection of fetal DNA in the blood, serum, or plasma ofa pregnant female, which contains both maternal DNA and fetal DNA. Thematernal DNA and fetal DNA may be present in the blood, serum or plasmaat ratios such as 97:3; however, the above-described method can be usedto detect the fetal DNA. This method of labeling can be used to detecttwo, three, four or more than four different genetic signals in thesample population

This method of labeling is especially useful for the detection of amutant allele that is among a large population of wild type alleles.Furthermore, this method of labeling allows the detection of a singlemutant cell in a large population of wild type cells. For example, thismethod of labeling can be used to detect a single cancerous cell among alarge population of normal cells. Typically, cancerous cells havemutations in the DNA sequence. The mutant DNA sequence can be identifiedeven if there is a large background of wild type DNA sequence. Thismethod of labeling can be used to screen, detect, or diagnosis any typeof cancer including but not limited to colon, renal, breast, bladder,liver, kidney, brain, lung, prostate, and cancers of the blood includingleukemia.

This labeling method can also be used to detect pathogenic organisms,including but not limited to bacteria, fungi, viruses, protozoa, andmycobacteria. It can also be used to discriminate between pathogenicstrains of microorganism and non-pathogenic strains of microorganismsincluding but not limited to bacteria, fungi, viruses, protozoa, andmycobacteria.

For example, there are several strains of Escherichia coii (E. coli),and most are non-pathogenic. However, several strains, such as E. coli O157 are pathogenic. There are genetic differences between non-pathogenicE. coli strains and pathogenic E. coli. The above described method oflabeling can be used to detect pathogenic microorganisms in a largepopulation of non-pathogenic organisms, which are sometimes associatedwith the normal flora of an individual.

VI. Analysis of the Locus of Interest

The loci of interest can be analyzed by a variety of methods includingbut not limited to fluorescence detection, DNA sequencing gel, capillaryelectrophoresis on an automated DNA sequencing machine, (e.g. the ABIPrism 3100 Genetic Analyzer or the ABI Prism 3700 Genetic Analyzer),microchannel electrophoresis, and other methods of sequencing, Sangerdideoxy sequencing, mass spectrometry, time of flight mass spectrometry,quadrupole mass spectrometry, magnetic sector mass spectrometry,electric sector mass spectrometry infrared spectrometry, ultravioletspectrometry, palentiostatic amperometry or by DNA hybridizationtechniques including Southern Blot, Slot Blot, Dot Blot, and DNAmicroarray, wherein DNA fragments would be useful as both “probes” and“targets,” ELISA, fluorimetry, fluorescence polarization, FluorescenceResonance Energy Transfer (FRET), SNP-IT, GeneChips, HuSNP, BeadArray,TaqMan assay, Invader assay, MassExtend, or MassCleave™ (hMC) method.

The loci of interest can be analyzed using gel electrophoresis followedby fluorescence detection of the incorporated nucleotide. Another methodto analyze or read the loci of interest is to use a fluorescent platereader or fluorimeter directly on the 96-well streptavidin coatedplates. The plate can be placed onto a fluorescent plate reader orscanner such as the Pharmacia 9200 Typhoon to read each locus ofinterest.

Alternatively, the PCR products of the loci of interest can be pooledand after “filling in” (FIG. 10), the products can be separated by size,using any method appropriate for the same, and then analyzed using avariety of techniques including but not limited to fluorescencedetection, DNA sequencing gel, capillary electrophoresis on an automatedDNA sequencing machine, microchannel electrophoresis, other methods ofsequencing, Sanger dideoxy sequencing, DNA hybridization techniquesincluding Southern Blot, Slot Blot, Dot Blot, and DNA microarray, massspectrometry, time of flight mass spectrometry, quadrupole massspectrometry, magnetic sector mass spectrometry, electric sector massspectrometry infrared spectrometry, ultraviolet spectrometry,palentiostatic amperometry. For example, polyacrylamide gelelectrophoresis can be used to separate DNA by size and the gel can bescanned to determine the color of fluorescence in each band (using e.g.,ABI 377 DNA sequencing machine or a Pharmacia Typhoon 9200).

In another embodiment, the sequence of the locus of interest can bedetermined by detecting the incorporation of a nucleotide that is 3′ tothe locus of interest, wherein said nucleotide is a different nucleotidefrom the possible nucleotides at the locus of interest. This embodimentis especially useful for the sequencing and detection of SNPs. Theefficiency and rate at which DNA polymerases incorporate nucleotidesvaries for each nucleotide.

According to the data from the Human Genome Project, 99% of all SNPs arebinary. The sequence of the human genome can be used to determine anucleotide that is 3′ to the SNP of interest. When a nucleotide that is3′ to the SNP site differs from the possible nucleotides at the SNPsite, a nucleotide that is one or more than one base 3′ to the SNP canbe used to determine the sequence of the SNP site.

For example, suppose the sequence of SNP X on chromosome 13 is to bedetermined. The sequence of the human genome indicates that SNP X caneither be adenosine or guanine and that a nucleotide 3′ to the locus ofinterest is a thymidine. A primer that contains a restriction enzymerecognition site for BsmF I, which is designed to be 13 bases from thelocus of interest after amplification, is used to amplify a DNA fragmentcontaining SNP X. Digestion with the restriction enzyme BsmF I generatesa 5′ overhang that contains the locus of interest, which can either beadenosine or guanine. The digestion products can be split into two “fillin” reactions: one contains dTTP, and the other reaction contains dCTP.If the locus of interest is homozygous for guanine, only the DNAmolecules that were mixed with dCTP will be filled in. If the locus ofinterest is homozygous for adenosine, only the DNA molecules that weremixed with dTTP will be filled in. If the locus of interest isheterozygous, the DNA molecules that were mixed with dCTP will be filledin as well as the DNA molecules that were mixed with dTTP. After washingto remove the excess dNTP, the samples are filled in with labeled ddATP,which is complimentary to the nucleotide (thymidine) that is 3′ to thelocus of interest. The DNA molecules that were filled in by the previousreaction will be filled in with labeled ddATP. If the individual ishomozygous for adenosine, the DNA molecules that were mixed with dTTPsubsequently will be filled in with the labeled ddATP. However, the DNAmolecules that were mixed with dCTP, would not have incorporated thatnucleotide, and therefore, could not incorporate the ddATP. Detection oflabeled ddATP only in the molecules that were mixed with dTTP indicatesthat the nucleotide at SNP X on chromosome 13 is adenosine.

In another embodiment, large scale screening for the presence or absenceof single nucleotide polymorphisms or mutations can be performed. One totens to hundreds to thousands of loci of interest on a single chromosomeor on multiple chromosomes can be amplified with primers as describedabove in the “Primer Design” section. The primers can be designed sothat each amplified loci of interest is of a different size (FIG. 2).The multiple loci of interest can be of a DNA sample from one individualrepresenting multiple loci of interest on a single chromosome, multiplechromosomes, multiple genes, a single gene, or any combination thereof.

When human data is being analyzed, the known sequence can be a specificsequence that has been determined from one individual (including e.g.the individual whose DNA is currently being analyzed), or it can be aconsensus sequence such as that published as part of the human genome.

Ratio of Alleles at Heterozygous Locus of Interest

In one embodiment, the ratio of alleles at a heterozygous locus ofinterest can be calculated. The intensity of a nucleotide at the loci ofinterest can be quantified using any number of computer programsincluding but not limited to GeneScan and ImageQuant. For example, for aheterozygous SNP, there are two nucleotides, and each should be presentin a 1:1 ratio. In a preferred embodiment, the ratio of multipleheterozygous SNPs can be calculated.

In one embodiment, the ratio for a variable nucleotide at alleles at aheterozygous locus of interest can be calculated. The intensity of eachvariable nucleotide present at the loci of interest can be quantifiedusing any number of computer programs including but not limited toGeneScan and ImageQuant. For example, for a heterozygous SNP, there willbe two nucleotides present, and each may be present in a 1:1 ratio. In apreferred embodiment, the ratio of multiple heterozygous SNPs can becalculated.

In another embodiment, the ratio of alleles at a heterozygous locus ofinterest on a chromosome is summed and compared to the ratio of allelesat a heterozygous locus of interest on a different chromosome. In apreferred embodiment, the ratio of alleles at multiple heterozygous lociof interest on a chromosome is summed and compared to the ratio ofalleles at multiple heterozygous loci of interest on a differentchromosome. The ratio obtained from SNP 1, SNP 2, SNP 3, SNP 4, etc onchromosome 1 can be summed. This ratio can then be compared to the ratioobtained from SNP A, SNP B, SNP C, SNP D, etc.

For example, 100 SNPs can be analyzed on chromosome 1. Of these 100SNPs, assume 50 are heterozygous. The ratio of the alleles atheterozygous SNPs on chromosome 1 can be summed, and should give a ratioof approximately 50:50. Likewise, of 100 SNPs analyzed on chromosome 21,assume 50 are heterozygous. The ratio of alleles at heterozygous SNPs onchromosome 21 is summed. With a normal number of chromosomes, the ratioshould be approximately 50:50, and thus there should be no differencebetween the ratio obtained from chromosome 1 and 21. However, if thereis an additional copy of chromosome 21, an additional allele will beprovided, and the ratio should be approximately 66:33. Thus, the ratiofor nucleotides at heterozygous SNPs can be used to detect the presenceor absence of chromosomal abnormalities. Any chromosomal abnormality canbe detected including aneuploidy, polyploidy, inversion, a trisomy, amonosomy, duplication, deletion, deletion of a part of a chromosome,addition, addition of a part of chromosome, insertion, a fragment of achromosome, a region of a chromosome, chromosomal rearrangement, andtranslocation. The method is especially useful for the detection oftrisomy 13, trisomy 18, trisomy 21, XXY, and XYY.

The present invention provides a method to quantitate a ratio for thealleles at a heterozygous locus of interest. The loci of interestinclude but are not limited to single nucleotide polymorphisms,mutations. There is no need to amplify the entire sequence of a gene orto quantitate the amount of a particular gene product. The presentinvention does not rely on quantitative PCR.

Detection of Fetal Chromosomal Abnormalities

As discussed above in the section entitled “DNA template,” the templateDNA can be obtained from a sample of a pregnant female, wherein thetemplate DNA comprises maternal template DNA and fetal template DNA. Inone embodiment, the template DNA is obtained from the blood of apregnant female. In a preferred embodiment, the template DNA is obtainedfrom the plasma or serum from the blood of a pregnant female.

In one embodiment, the template DNA from the sample from the pregnantfemale comprises both maternal template DNA and fetal template DNA. Inanother embodiment, maternal template DNA is obtained from any nucleicacid containing source including but not limited to cell, tissue, blood,serum, plasma, saliva, urine, tears, vaginal secretion, lymph fluid,cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascitic fluid,fecal matter, or body exudates, and sequenced to identify homozygous orheterozygous loci of interest, which are the loci of interest analyzedon the template DNA obtained from the sample from the pregnant female.

In a preferred embodiment, the sequence of the alleles of multiple lociof interest on maternal template DNA is determined to identifyhomozygous loci of interest. In another embodiment, the sequence of thealleles of multiple loci of interest on maternal template DNA isdetermined to identify heterozygous loci of interest. The sequence ofthe alleles of multiple loci of interest on maternal template DNA can bedetermined in a single reaction or in multiple reactions.

For example, if 100 maternal loci of interest on chromosome 21 and 100maternal loci of interest on chromosome 1 are analyzed, one wouldpredict approximately 50 loci of interest on each chromosome to behomozygous and 50 to be heterozygous. The 50 homozygous loci ofinterest, or the 50 heterozygous loci of interest or the 50 homozygousand 50 heterozygous loci of interest, or any combination of thehomozygous and heterozygous loci or interest on each chromosome can beanalyzed using the template DNA from the sample from the pregnantfemale.

The locus of interest on the template DNA from the sample of thepregnant female is analyzed using the amplification, isolation,digestion, fill in, and detection methods described above. The sameprimers used to analyze the locus of interest on the maternal templateDNA are used to screen the template DNA from the sample from thepregnant female. Any number of loci of interest can be analyzed on thetemplate DNA from the sample from the pregnant female. For example, 1,1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-100, 100-150, 150-200, 200-250, 250-300, 300-500, 500-1000,1000-2000, 2000-3000, 3000-4000 or more than 4000 homozygous maternalloci of interest can be analyzed in the template DNA from the samplefrom the pregnant female. In a preferred embodiment, multiple loci ofinterest on multiple chromosomes are analyzed.

From the population of homozygous maternal loci of interest, there willbe both heterozygous and homozygous loci of interest from the templateDNA from the sample from the pregnant female; the heterozygous loci ofinterest can be further analyzed. At heterozygous loci of interest, theratio of alleles can be used to determine the number of chromosomes thatare present.

The percentage of fetal DNA present in the sample from the pregnantfemale can be calculated by determining the ratio of alleles at aheterozygous locus of interest on a chromosome that is not typicallyassociated with a chromosomal abnormality. In a preferred embodiment,the ratio of alleles at multiple heterozygous loci of interest on achromosome can be used to determine the percentage of fetal DNA. Forexample, chromosome 1, which is the largest chromosome in the humangenome, can be used to determine the percentage of fetal DNA.

For example, suppose SNP X is homozygous at the maternal template DNA(A/A). At SNP X, the template DNA from the sample from the pregnantfemale, which can contain both fetal DNA and maternal DNA, isheterozygous (A/G). The nucleotide guanine represents the fetal DNAbecause at SNP X the mother is homozygous, and thus the guanine isattributed to the fetal DNA. The guanine at SNP X can be used tocalculate the percentage of fetal DNA in the sample.

Alternatively, multiple loci of interest on two or more chromosomes canbe examined to determine the percentage of fetal DNA. For example,multiple loci of interest can be examined on chromosomes 13, and 18 todetermine the percentage of fetal DNA because organisms with chromosomalabnormalities at chromosome 13 and 18 are not viable.

Alternatively, for a male fetus, a marker on the Y chromosome can beused to determine the amount of fetal DNA present in the sample. A panelof serial dilutions can be made using the template DNA isolated from thesample from the pregnant female, and quantitative PCR analysisperformed. Two PCR reactions can be performed: one PCR reaction toamplify a marker on the Y chromosome, for example SRY, and the otherreaction to amplify a region on any of the autosomal chromosomes. Theamount of fetal DNA can be calculated using the following formula:

Percent Fetal DNA: (last dilution Y chromosome detected/last dilutionautosomal chromosome detected)*2*100.

If at SNP A, the mother is homozygous A/A, and the fetus is heterozygousA/G, then the ratio of A:G can be used to detect chromosomalabnormalities. If the fetal DNA is fifty percent (50%) of the DNA in thematernal blood, then at SNP A where the maternal nucleotide is anadenine and the other nucleotide is a guanine, one would expect theratio of adenine (two adenines from the maternal template DNA and onefrom the fetal template DNA) to guanine (from the fetal template DNA) tobe 25:75 or 0.33. However, if the fetus has a trisomy of this particularchromosome, and the additional chromosome is contributed by the mother,and thus an additional adenine nucleotide is present, then one wouldexpect the ratio of 0.25 (50 (G)/(2*50 maternal A+2*50 fetal A). Thus,there is a difference of 8% between the ratio obtained from a chromosomepresent in two copies, and a chromosome present in a trisomy condition.On the other hand, if the additional chromosome is contributed by thefather, and thus, an additional guanine is present, then one wouldexpect the ratio of 0.66 (2*50 for G fetal allele/(2*50 maternal Aallele+50 for fetal A allele).

However, if the fetal DNA is 40% of the DNA in the maternal blood, theexpected ratio without a trisomy is 0.25 (40 for fetal G allele/2*60 formaternal A allele+1*60 for fetal A allele). If the fetus has a trisomy,and the additional chromosome is provided by the mother, the expectedratio would be 0.20 (40 for fetal G allele/(2*60 for maternal Aallele+2*40 for fetal A allele). A 5% difference between the ratiosobtained from a chromosome present in two copies and a chromosomepresent in the Trisomy condition is detected.

In another embodiment, multiple loci of interest on multiple chromosomescan be examined. The ratios for the alleles at each heterozygous locusof interest on a chromosome can be summed and compared to the ratios forthe alleles at each locus of interest on a different chromosome. Thechromosomes that are compared can be of human origin, and include butare not limited to chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, and Y. The ratio obtainedfrom multiple chromosomes can be compared to the ratio obtained for asingle chromosome or from multiple chromosomes.

In one embodiment, one of the chromosomes used in the comparison can bechromosome 13, 15, 16; 18, 21, 22, X or Y. In a preferred embodiment,the ratios on chromosomes 13, 18, and 21 are compared.

For example, assuming 40% fetal DNA in the sample from the pregnantfemale, the ratio of the alleles at a heterozygous locus of interest onchromosome 1 will be 0.25 (40 for fetal G allele/(2*60 for maternal Aallele+40 for fetal A allele). Likewise, the ratio of alleles at aheterozygous locus of interest on chromosome 21 will be present in aratio of 0.25. However, in a fetus with trisomy 21 where the additionalchromosome is contributed by the mother, the nucleotides at aheterozygous locus of interest on chromosome 21 will be present in aratio of 0.20 (40 for fetal G allele/(60*2 for maternal A allele+40*2for fetal A allele). By contrast, the ratio for chromosome 1 will remainat 0.25, and thus the 5% difference in ratios will signify an additionalchromosome. One to tens to hundreds to thousands of loci of interest canbe analyzed.

In another embodiment, the loci of interest on the template DNA from thesample from the pregnant female can be genotyped without prioridentification of the homozygous maternal loci of interest. It is notnecessary to genotype the maternal template DNA prior to analysis of thetemplate DNA containing both maternal and fetal template. DNA.

The ratio of the alleles at the loci of interest can be used todetermine the presence or absence of a chromosomal abnormality. Thetemplate DNA from the sample from the pregnant female contains bothmaternal template DNA and fetal template DNA. There are 3 possibilitiesat each SNP for either the maternal template DNA or the fetal templateDNA: heterozygous, homozygous for allele 1, or homozygous for allele 2.The possible nucleotide ratios for a SNP that is either an adenine or aguanine are shown in Table II. The ratios presented in Table II arecalculated with the fetal DNA at 50% of the DNA in the sample from thepregnant female.

TABLE II Ratios for nucleotides for a heterozygous SNP. Fetal SNPMaternal SNP A/A G/G A/G A/A 100% A N/A 75% A, 25% G G/G N/A 100% G 25%A, 75% G A/G 75% A, 25% G 25% A, 75% G 50% A, 50% G

There are three nucleotide ratios: 100% of a single nucleotide, 50:50,or 75:25. These ratios will vary depending on the amount of fetal DNApresent in sample from the pregnant female. However, the percentage offetal DNA should be constant regardless of the chromosome analyzed.Therefore, if chromosomes are present in two copies, the abovecalculated ratios will be seen.

On the other hand, these percentages will vary when an additionalchromosome is present. For example, assume that SNP X can be adenine orguanine, and that the percentage of fetal DNA in the sample from thepregnant female is 50%. Analysis of the loci of interest on chromosome 1will provide the ratios discussed above: 100:0, 50:50, and 75:25. Thepossible ratios for a SNP that is A/G with an additional chromosome areprovided in Table III.

TABLE III Nucleotides ratios at a SNP when an additional copy of achromosome is present Maternal Fetal SNP SNPX A/A/A G/G/G A/G/G A/A/GA/A 100% A N/A 60% A, 40% G 80% A, 20% G G/G N/A 100% G 20% A, 80% G 40%A, 60% G A/G  80% A, 20% G  20% A, 80% G 40% A, 60% G 60% A, 40% G

The possible ratios for the alleles at a heterozygous SNP with anadditional copy of a chromosome are: 0:100, 40:60, and 20:80. Two ofthese ratios, 40:60, and 20:80 differ from the ratios of alleles atheterozygous SNPs obtained with two copies of a chromosome. As discussedabove, the ratios for the nucleotides at a heterozygous SNP depend onthe amount of fetal DNA present in the sample. However, the ratios,whatever they are, will remain constant across chromosomes unless thereis a chromosomal abnormality.

The ratio of alleles at heterozygous loci of interest on a chromosomecan be compared to the ratio for alleles at heterozygous loci ofinterest on a different chromosome. For example, the ratio for multipleloci of interest on chromosome 1 (the ratio at SNP 1, SNP 2, SNP 3, SNP4, etc.) can be compared to the ratio for multiple loci of interest onchromosome 21 (the ratio at SNP A, SNP B, SNP C, SNP D, etc.). Anychromosome can be compared to any other chromosome. There is no limit tothe number of chromosomes that can be compared.

Referring back to the data in Tables II and III, the ratios fornucleotides at a heterozygous SNP on chromosome 1, which was present intwo copies, were 25:75, and 50:50. On the other, the ratio fornucleotides at a heterozygous SNP on chromosome 21, which was present inthree copies, were 40:60, and 20:80. The difference between these tworatios indicates a chromosomal abnormality. The ratios can bepre-calculated for the full range of varying degrees of fetal DNApresent in the maternal serum. Tables II and III demonstrate that bothmaternal homozygous and heterozygous loci of interest can be used todetect the presence of a fetal chromosomal abnormality.

The above example illustrates how the ratios for nucleotides atheterozygous SNPs can be used to detect the presence of an additionalchromosome. The same type of analysis can be used to detect chromosomalrearrangements, translocations, mini-chromosomes, duplications ofregions of chromosomes, monosomies, deletions of regions of chromosomes,and fragments of chromosomes. The method does not require genotyping ofthe mother or the father, however, it may be done to reduce the numberof SNPs that need to be analyzed with the plasma sample.

The present invention does not quantitate the amount of a fetal geneproduct, nor is the utility of the present invention limited to theanalysis of genes found on the Y chromosome. The present invention doesnot merely rely on the detection of a paternally inherited nucleic acid,rather, the present invention provides a method that allows the ratio ofmaternal to fetal alleles at loci of interest, including SNPs, to becalculated.

In another embodiment, a single allele at a locus of interest can beused to determine the presence or absence of a chromosomal abnormalityand detect a genetic disorder in the fetus. In a preferred embodiment,the maternal allele at a locus of interest is used to determine thepresence or absence of a chromosomal abnormality in the fetus. Thebiological mother can be genotyped to identify a homozygous locus ofinterest. Likewise, the biological father can be genotyped to identify ahomozygous locus of interest. The locus of interest wherein the maternaltemplate DNA is homozygous for one allele and the paternal template DNAis homozygous for the other allele is analyzed using the template DNAobtained from the plasma of the mother, which contains both maternal andfetal template DNA. Any number of loci of interest can be analyzedincluding but not limited to 1, 1-5, 5-10, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, 100-150, 150-200, 200-250, 250-300,300-500, 500-1000, 1000-2000, 2000-3000, 3000-4000, 4000-8000,8000-16000, 16000-32000 or greater than 32000 loci of interest.

In a preferred embodiment, the signal from the maternal genome and thefetal allele, which was inherited from the mother, at the locus ofinterest is quantitated. For example, if the 5′ overhang, which isgenerated after digestion with the type IIS enzyme, is filled in with anucleotide that is fluorescently labeled, the intensity of theincorporated dye can be quantitated

Maternal Template DNA—Homozygous for Adenine

Allele 1 5′ CCG A* 3′ GGC T G T G Overhang position 1 2 3 4 Allele 25′ CCG A* 3′ GGC T G T G Overhang position 1 2 3 4

Paternal Template DNA—Homozygous for Cytosine

Allele 1 5′ CCG C* 3′ GGC G G T G Overhang position 1 2 3 4 Allele 25′ CCG C* 3′ GGC T G T G Overhang position 1 2 3 4

Template DNA in the Plasma—Both Maternal Template DNA and Fetal TemplateDNA

-   -   Maternal Template DNA—Homozygous for Adenine

Allele 1 5′ CCG A* 3′ GGC T G T G Overhang position 1 2 3 4 Allele 25′ CCG A* 3′ GGC T G T G Overhang position 1 2 3 4

Fetal Template DNA—Heterozygous

Allele 1 5′ CCG A* 3′ GGC T G T G Overhang position 1 2 3 4 Allele 25′ CCG ddC 3′ GGC T G T G Overhang position 1 2 3 4

The template DNA obtained from the plasma of the pregnant female isfilled in with labeled ddATP, and unlabeled ddCTP (depicted as ddCabove), ddGTP, and ddTTP. The plasma DNA contains two maternal adeninealleles, and one fetal adenine allele. By filling in with labeled ddATPand unlabeled ddCTP, only the maternal allele and the fetal alleleinherited from the mother are detected. The paternal allele is notdetected in this manner. The fill-in reactions can be performed asdescribed in the Examples below.

A single locus of interest can be analyzed or multiple loci of interest.The intensity of the maternal allele at multiple loci of interest can bequantitated. An average can be calculated for a chromosome and comparedto the average obtained for a different chromosome. For example, theaverage intensity of the maternal allele and the fetal allele inheritedfrom the mother at chromosome 1 can be compared to the average intensityof the maternal allele and the fetal allele inherited from the mother atchromosomes 13, 18, or 21. In a preferred embodiment, chromosomes 13,15, 18, 21, 22, X and Y, when applicable, are compared.

The signal from a locus of interest may be stronger than another locusof interest. However, there is no reason why the signal from the locusof interest on one chromosome would be stronger than the signal from thelocus of interest on another chromosome. While the signal from variousloci of interest may be variable, the variation should be seen acrossthe genome. The average signal of the loci of interest should be thesame when any chromosomes are compared.

The conditions of the PCR reaction can be optimized so that anequivalent amount of PCR product is produced. For example, theconcentration of the primers, the concentration of nucleotides, and thenumber of cycles for each loci of interest can be optimized. Inaddition, the fill-in reactions can be done under conditions such thatany increase in a specific allele can be detected. The fill-in reactionconditions can be optimized to detect any increase in the allele ofinterest including but not limited to the concentration of reagents, thetime of the fill-in reaction, and the temperature of the reaction.

With a normal genetic karyotype, the signal at each locus of interestcomprise signal from the maternal genome, and signal from the fetalallele, which was inherited from the mother. The percent of fetal DNA inthe sample remains constant, regardless of the chromosome that isanalyzed. For example, if at SNP X, the maternal genome is A/A, and thepaternal genome is G/G, then the fetal genome will be A/G, and the fetaladenine allele will comprise a specified percentage of the signal fromthe adenine allele. If the percentage of fetal DNA is 20% in thematernal plasma, then the fetal adenine allele will contribute 20% ofthe signal for the adenine allele. The contribution of the fetal allele,which was inherited from the mother, will be constant for any locus ofinterest that is analyzed.

When there is a chromosomal abnormality, the signal from the maternalgenome and the fetal allele, which was inherited from the mother, at theloci of interest will differ from the signal observed for otherchromosomes. For example, with a Trisomy, the signal at the locus ofinterest will comprise the maternal genome and two fetal alleles, whichwere inherited from the mother. The signal from the loci of interest forthe chromosome that is present in three copies will have thecontribution of an additional fetal allele, which will alter the signalof the alleles at these loci of interest.

In another embodiment, a ratio can be calculated using a single alleleand a standard DNA of known quantity. In a preferred embodiment, a ratiois calculated using the alleles of the maternal genome, and the fetalallele, which was inherited from the mother, and a standard DNA. Thebiological mother can be genotyped to identify a homozygous locus ofinterest. Likewise, the biological father can be genotyped to identify ahomozygous locus of interest. The locus of interest wherein the maternaltemplate DNA is homozygous for one allele and the paternal template DNAis homozygous for the other allele is analyzed using the template DNAobtained from the plasma of the mother, which contains both maternal andfetal template DNA.

In a preferred embodiment, the signal from the maternal genome and thefetal allele, which was inherited from the mother, at the locus ofinterest is quantitated. For example, if the 5′ overhang, which isgenerated after digestion with the type IIS enzyme, is filled in with anucleotide that is fluorescently labeled, the intensity of theincorporated dye can be quantitated.

Template DNA in the Plasma—Both Maternal Template DNA and Fetal TemplateDNA

-   -   Maternal Template DNA—Homozygous for Adenine

Allele 1 5′ CCG A* 3′ GGC T G T G Overhang position 1 2 3 4 Allele 25′ CCG A* 3′ GGC T G T G Overhang position 1 2 3 4

Fetal Template DNA—Heterozygous

Allele 1 5′ CCG A* 3′ GGC T G T G Overhang position 1 2 3 4 Allele 25′ CCG ddC 3′ GGC T G T G Overhang position 1 2 3 4

The template DNA obtained from the plasma of the pregnant female isfilled in with labeled ddATP, and unlabeled ddCTP (depicted as ddCabove), ddGTP, and ddTTP. The plasma DNA contains two maternal adeninealleles, and one fetal adenine allele. By filling in with labeled ddATPand unlabeled ddCTP, only the maternal allele and the fetal alleleinherited from the mother are detected.

A single locus of interest or multiple loci of interest can be analyzed.For each locus of interest, a DNA molecule is designed to migrate atabout the same position as the locus of interest. In a preferredembodiment, the DNA molecule is of known quantity. A ratio is calculatedusing the alleles of the maternal genome and the fetal allele, which wasinherited from the mother, and the DNA molecule designed to migrate atabout the same position as the locus of interest. For example, if thelocus of interest is designed to migrate at 30 base pairs, the DNAmolecule can be designed to migrate at about 30 base pairs including butnot limited to 20-25, 25-30, 30-35, 35-45, and greater than 45. Thealleles of the maternal genome and the fetal allele, which was inheritedfrom the mother, and the standard DNA molecule can be analyzed in thesame reaction or can be analyzed in a separate reaction. The alleles ofthe maternal genome and the fetal allele, which was inherited from themother, and the standard DNA molecule can be analyzed in the same laneof a gel or can be analyzed in separate lanes of a gel. The use ofstandard DNA molecules of known quantity, which are designed to migrateat the same position as the loci of interest, will correct for variousfactors including but not limited to the intensity of the bands relativeto the location on the gel.

The ratio of multiple loci of interest on a chromosome can bequantitated, and an average calculated. The average can be compared tothe average obtained for another chromosome. The ratio is used toindicate the presence or absence of a chromosomal abnormality. Analysisof the alleles of the maternal genome and the fetal allele also allowsdetection of single gene or multi-gene genetic disorders.

Any chromosome of any organism can be analyzed using the methods of theinvention. For example, in humans, chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X or Y can beanalyzed using the methods of the invention. The ratio for the allelesat a heterozygous locus of interest on any chromosome can be compared tothe ratio for the alleles at a heterozygous locus of interest on anyother chromosome.

Thus, the present invention provides a non-invasive technique, which isindependent of fetal cell isolation, for rapid, accurate and definitivedetection of chromosome abnormalities in a fetus. The present inventionalso provides a non-invasive method for determining the sequence of DNAfrom a fetus. The present invention can be used to detect anyalternation in gene sequence as compared to the wild type sequenceincluding but not limited to point mutation, reading frame shift,transition, transversion, addition, insertion, deletion,addition-deletion, frame-shift, missense, reverse mutation, andmicrosatellite alteration.

Detection of Fetal Chromosomal Abnormalities Using Short Tandem Repeats

Short tandem repeats (STRs) are short sequences of DNA, normally of 2-5base pairs in length, which are repeated numerous times in a head-tailmanner. Tandemly repeated DNA sequences are widespread throughout thehuman genome, and show sufficient variability among the individuals in apopulation. Minisatellites have core repeats with 9-80 base pairs.

In another embodiment, short tandem repeats can be used to detect fetalchromosomal abnormalities. Template DNA can be obtained from a nucleicacid containing sample including but not limited to cell, tissue, blood,serum, plasma, saliva, urine, tears, vaginal secretion, lymph fluid,cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascitic fluid,fecal matter, or body exudates. In another embodiment, a cell lysisinhibitor is added to the nucleic acid containing sample. In a preferredembodiment, the template DNA is obtained from the blood of a pregnantfemale. In another embodiment, the template DNA is obtained from theplasma or serum from the blood of a pregnant female.

The template DNA obtained from the blood of the pregnant female willcontain both fetal DNA and maternal DNA. The fetal DNA comprises STRsfrom the mother and the father. The variation in the STRs between themother and father can be used to detect chromosomal abnormalities.

Primers can be designed to amplify short tandem repeats. Any method ofamplification can be used including but not limited to polymerase chainreaction, self-sustained sequence reaction, ligase chain reaction, rapidamplification of cDNA ends, polymerase chain reaction and ligase chainreaction, Q-beta phage amplification, strand displacement amplification,and splice overlap extension polymerase chain reaction. In a preferredembodiment, PCR is used.

Any number of short tandem repeats can be analyzed including but notlimited to 1-5, 5-10, 10-50, 50-100, 100-200, 200-300, 300-400, 400-500,500-1000, and greater than 1000. The short tandem repeats can beanalyzed in a single PCR reaction or in multiple PCR reactions. In apreferred embodiment, STRs from multiple chromosomes are analyzed.

After amplification, the PCR products can be analyzed by any number ofmethods including but not restricted to get electrophoresis, and massspectrometry. The template DNA from the pregnant female comprises STRsof maternal and paternal origin. The STRs of paternal origin representthe fetal DNA. The paternal and maternal STRs may be identical in lengthor the maternal and the paternal STRs may differ.

Heterozygous STRs are those of which the maternal and paternal differ inlength. The amount of each PCR product can be quantitated for eachheterozygous STR. With a normal number of chromosomes, the amount ofeach PCR product should be approximately equal. However, with an extrachromosome, one of the STR PCR products will be present at a greateramount.

For example, multiple STRs on chromosome 1 can be analyzed on thetemplate DNA obtained from the blood of the pregnant female. Each STR,whether of maternal or paternal origin, should be present atapproximately the same amount. Likewise, with two chromosome 21s, eachSTR should be present at approximately the same amount. However, with atrisomy 21, one of the STR PCR products, when the maternal and paternaldiffer in length (a heterozygous STR) should be present at a higheramount. The ratio for each heterozygous STR on one chromosome can becompared to the ratio for each heterozygous STR on a differentchromosome, wherein a difference indicates the presence or absence of achromosomal abnormality.

Kits

The methods of the invention are most conveniently practiced byproviding the reagents used in the methods in the form of kits. A kitpreferably contains one or more of the following components: writteninstructions for the use of the kit, appropriate buffers, salts, DNAextraction detergents, primers, nucleotides, labeled nucleotides, 5′ endmodification materials, and if desired, water of the appropriate purity,confined in separate containers or packages, such components allowingthe user of the kit to extract the appropriate nucleic acid sample, andanalyze the same according to the methods of the invention. The primersthat are provided with the kit will vary, depending upon the purpose ofthe kit and the DNA that is desired to be tested using the kit.

A kit can also be designed to detect a desired or variety of singlenucleotide polymorphisms, especially those associated with an undesiredcondition or disease. For example, one kit can comprise, among othercomponents, a set or sets of primers to amplify one or more loci ofinterest associated with Huntington's disease. Another kit can comprise,among other components, a set or sets of primers for genes associatedwith a predisposition to develop type I or type II diabetes. Still,another kit can comprise, among other components, a set or sets ofprimers for genes associated with a predisposition to develop heartdisease. Details of utilities for such kits are provided in the“Utilities” section below.

Utilities

The methods of the invention can be used whenever it is desired to knowthe genotype of an individual. The method of the invention is especiallyuseful for the detection of genetic disorders. The method of theinvention is especially useful as a non-invasive technique for thedetection of genetic disorders in a fetus. In a preferred embodiment,the method of the invention provides a method for identification ofsingle nucleotide polymorphisms.

In a preferred embodiment, the method is useful for detectingchromosomal abnormalities including but not limited to trisomies,monosomies, duplications, deletions, additions, chromosomalrearrangements, translocations, and other aneuploidies. The method isespecially useful for the detection of chromosomal abnormalities in afetus.

In a preferred embodiment, the method of the invention provides a methodfor identification of the presence of a disease in a fetus, especially agenetic disease that arises as a result of the presence of a genomicsequence, or other biological condition that it is desired to identifyin an individual for which it is desired to know the same. Theidentification of such sequence in the fetus based on the presence ofsuch genomic sequence can be used, for example, to determine if thefetus is a carrier or to assess if the fetus is predisposed todeveloping a certain genetic trait, condition or disease. The method ofthe invention is especially useful in prenatal genetic testing ofparents and child.

Examples of diseases that can be diagnosed by this invention are listedin Table IV.

TABLE IV Achondroplasia Adrenoleukodystrophy, X-LinkedAgammaglobulinemia, X-Linked Alagille Syndrome Alpha-ThalassemiaX-Linked Mental Retardation Syndrome Alzheimer Disease AlzheimerDisease, Early-Onset Familial Amyotrophic Lateral Sclerosis OverviewAndrogen Insensitivity Syndrome Angelman Syndrome Ataxia Overview,Hereditary Ataxia-Telangiectasia Becker Muscular Dystrophy also TheDystrophinopathies) Beckwith-Wiedemann Syndrome Beta-ThalassemiaBiotinidase Deficiency Branchiootorenal Syndrome BRCA1 and BRCA2Hereditary Breast/Ovarian Cancer Breast Cancer CADASIL Canavan DiseaseCancer Charcot-Marie-Tooth Hereditary Neuropathy Charcot-Marie-ToothNeuropathy Type 1 Charcot-Marie-Tooth Neuropathy Type 2Charcot-Marie-Tooth Neuropathy Type 4 Charcot-Marie-Tooth NeuropathyType X Cockayne Syndrome Colon Cancer Contractural Arachnodactyly,Congenital Craniosynostosis Syndromes (FGFR-Related) Cystic FibrosisCystinosis Deafness and Hereditary Hearing Loss DRPLA(Dentatorubral-Pallidoluysian Atrophy) DiGeorge Syndrome (also 22q11Deletion Syndrome) Dilated Cardiomyopathy, X-Linked Down Syndrome(Trisomy 21) Duchenne Muscular Dystrophy (also The Dystrophinopathies)Dystonia, Early-Onset Primary (DYT1) Dystrophinopathies, TheEhlers-Danlos Syndrome, Kyphoscoliotic Form Ehlers-Danlos Syndrome,Vascular Type Epidermolysis Bullosa Simplex Exostoses, HereditaryMultiple Facioscapulohumeral Muscular Dystrophy Factor V LeidenThrombophilia Familial Adenomatous Polyposis (FAP) FamilialMediterranean Fever Fragile X Syndrome Friedreich Ataxia FrontotemporalDementia with Parkinsonism-17 Galactosemia Gaucher DiseaseHemochromatosis, Hereditary Hemophilia A Hemophilia B HemorrhagicTelangiectasia, Hereditary Hearing Loss and Deafness, Nonsyndromic, DFNA(Connexin 26) Hearing Loss and Deafness, Nonsyndromic, DFNB 1 (Connexin26) Hereditary Spastic Paraplegia Hermansky-Pudlak SyndromeHexasaminidase A Deficiency (also Tay-Sachs) Huntington DiseaseHypochondroplasia Ichthyosis, Congenital, Autosomal RecessiveIncontinentia Pigmenti Kennedy Disease (also Spinal and Bulbar MuscularAtrophy) Krabbe Disease Leber Hereditary Optic Neuropathy Lesch-NyhanSyndrome Leukemias Li-Fraumeni Syndrome Limb-Girdle Muscular DystrophyLipoprotein Lipase Deficiency, Familial Lissencephaly Marfan SyndromeMELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-LikeEpisodes) Monosomies Multiple Endocrine Neoplasia Type 2 MultipleExostoses, Hereditary Muscular Dystrophy, Congenital Myotonic DystrophyNephrogenic Diabetes Insipidus Neurofibromatosis 1 Neurofibromatosis 2Neuropathy with Liability to Pressure Palsies, Hereditary Niemann-PickDisease Type C Nijmegen Breakage Syndrome Norrie Disease OculocutaneousAlbinism Type 1 Oculopharyngeal Muscular Dystrophy Ovarian CancerPallister-Hall Syndrome Parkin Type of Juvenile Parkinson DiseasePelizaeus-Merzbacher Disease Pendred Syndrome Peutz-Jeghers SyndromePhenylalanine Hydroxylase Deficiency Prader-Willi Syndrome PROP1-Related Combined Pituitary Hormone Deficiency (CPHD) Prostate CancerRetinitis Pigmentosa Retinoblastoma Rothmund-Thomson SyndromeSmith-Lemli-Opitz Syndrome Spastic Paraplegia, Hereditary Spinal andBulbar Muscular Atrophy (also Kennedy Disease) Spinal Muscular AtrophySpinocerebellar Ataxia Type 1 Spinocerebellar Ataxia Type 2Spinocerebellar Ataxia Type 3 Spinocerebellar Ataxia Type 6Spinocerebellar Ataxia Type 7 Stickler Syndrome (HereditaryArthroophthalmopathy) Tay-Sachs (also GM2 Gangliosidoses) TrisomiesTuberous Sclerosis Complex Usher Syndrome Type I Usher Syndrome Type IIVelocardiofacial Syndrome (also 22q11 Deletion Syndrome) VonHippel-Lindau Syndrome Williams Syndrome Wilson Disease X-LinkedAdrenoleukodystrophy X-Linked Agammaglobulinemia X-Linked DilatedCardiomyopathy (also The Dystrophinopathies) X-Linked Hypotonic FaciesMental Retardation Syndrome

The method of the invention is useful for screening an individual atmultiple loci of interest, such as tens, hundreds, or even thousands ofloci of interest associated with a genetic trait or genetic disease bysequencing the loci of interest that are associated with the trait ordisease state, especially those most frequently associated with suchtrait or condition. The invention is useful for analyzing a particularset of diseases including but not limited to heart disease, cancer,endocrine disorders, immune disorders, neurological disorders,musculoskeletal disorders, ophthalmologic disorders, geneticabnormalities, trisomies, monosomies, transversions, translocations,skin disorders, and familial diseases.

The method of the invention can also be used to confirm or identify therelationship of a DNA of unknown sequence to a DNA of known origin orsequence, for example, for use in, maternity or paternity testing, andthe like.

Having now generally described the invention, the same will becomebetter understood by reference to certain specific examples which areincluded herein for purposes of illustration only and are not intendedto be limiting unless otherwise specified.

EXAMPLES

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the claims.

Example 1

DNA sequences were amplified by PCR, wherein the annealing step in cycle1 was performed at a specified temperature, and then increased in cycle2, and further increased in cycle 3 for the purpose of reducingnon-specific amplification. The TM1 of cycle 1 of PCR was determined bycalculating the melting temperature of the 3′ region, which anneals tothe template DNA, of the second primer. For example, in FIG. 1B, the TM1can be about the melting temperature of region “c.” The annealingtemperature was raised in cycle 2, to TM2, which was about the meltingtemperature of the 3′ region, which anneals to the template DNA, of thefirst primer. For example, in FIG. 1C, the annealing temperature (TM2)corresponds to the melting temperature of region “b.” In cycle 3, theannealing temperature was raised to TM3, which was about the meltingtemperature of the entire sequence of the second primer. For example, inFIG. 1D, the annealing temperature (TM3) corresponds to the meltingtemperature of region “c”+region “d”. The remaining cycles ofamplification were performed at TM3.

Preparation of Template DNA

The template DNA was prepared from a 5 ml sample of blood obtained byvenipuncture from a human volunteer with informed consent. Blood wascollected from 36 volunteers. Template DNA was isolated from each bloodsample using QIAmp DNA Blood Midi Kit supplied by QIAGEN (Catalog number51183). Following isolation, the template DNA from each of the 36volunteers was pooled for further analysis.

Primer Design

The following four single nucleotide polymorphisms were analyzed: SNPHC21S00340, identification number as assigned by Human Chromosome 21cSNP Database, (FIG. 3, lane 1) located on chromosome 21; SNP TSC0095512 (FIG. 3, lane 2) located on chromosome 1, SNP TSC 0214366 (FIG.3, lane 3) located on chromosome 1; and SNP TSC 0087315 (FIG. 3, lane 4)located on chromosome 1. The SNP Consortium Ltd database can be accessedat http://snp.cshl.org/, website address effective as of Feb. 14, 2002.

SNP HC21S00340 was amplified using the following primers:

First primer: (SEQ ID NO: 9) 5′TAGAATAGCACTGAATTCAGGAATACAATCATTGTCAC 3′Second primer: (SEQ ID NO: 10) 5′ATCACGATAAACGGCCAAACTCAGGTTA3′

SNP TSC0095512 was amplified using the following primers:

First primer: (SEQ ID NO: 11) 5′AAGTTTAGATCAGAATTCGTGAAAGCAGAAGTTGTCTG3′ Second primer: (SEQ ID NO: 12) 5′TCTCCAACTAACGGCTCATCGAGTAAAG 3′

SNP TSC0214366 was amplified using the following primers:

First primer: (SEQ ID NO: 13) 5′ATGACTAGCTATGAATTCGTTCAAGGTAGAAAATGGAA3′ Second primer: (SEQ ID NO: 14) 5′GAGAATTAGAACGGCCCAAATCCCACTC3′

SNP TSC 0087315 was amplified using the following primers:

First primer: (SEQ ID NO: 15) 5′TTACAATGCATGAATTCATCTTGGTCTCTCAAAGTGC 3′Second primer: (SEQ ID NO: 16) 5′TGGACCATAAACGGCCAAAAACTGTAAG 3′.

All primers were designed such that the 3′ region was complementary toeither the upstream or downstream sequence flanking each locus ofinterest and the 5′ region contained a restriction enzyme recognitionsite. The first primer contained a biotin tag at the 5′ end and arecognition site for the restriction enzyme EcoRI. The second primercontained the recognition site for the restriction enzyme BceA I.

PCR Reaction

All four loci of interest were amplified from the template genomic DNAusing PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202). The components ofthe PCR reaction were as follows: 40 ng of template DNA, 5 μM firstprimer, 5 μM second primer, 1× HotStarTaq Master Mix as obtained fromQiagen (Catalog No. 203443). The HotStarTaq Master Mix contained DNApolymerase, PCR buffer, 200 μM of each dNTP, and 1.5 mM MgCl₂.

Amplification of each template DNA that contained the SNP of interestwas performed using three different series of annealing temperatures,herein referred to as low stringency annealing temperature, mediumstringency annealing temperature, and high stringency annealingtemperature. Regardless of the annealing temperature protocol, each PCRreaction consisted of 40 cycles of amplification. PCR reactions wereperformed using the HotStarTaq Master Mix Kit supplied by QIAGEN. Asinstructed by the manufacturer, the reactions were incubated at 95° C.for 15 min. prior to the first cycle of PCR. The denaturation step aftereach extension step was performed at 95° C. for 30 sec. The annealingreaction was performed at a temperature that permitted efficientextension without any increase in temperature.

The low stringency annealing reaction comprised three differentannealing temperatures in each of the first three cycles. The annealingtemperature for the first cycle was 37° C. for 30 sec.; the annealingtemperature for the second cycle was 57° C. for 30 sec.; the annealingtemperature for the third cycle was 64° C. for 30 sec. Annealing wasperformed at 64° C. for subsequent cycles until completion.

As shown in the photograph of the gel (FIG. 3A), multiple bands wereobserved after amplification of SNP TSC 0087315 (lane 4). Amplificationof SNP HC21S00340 (lane 1), SNP TSC0095512 (lane 2), and SNP TSC0214366(lane 3) generated a single band of high intensity and one band of faintintensity, which was of higher molecular weight. When the low annealingtemperature conditions were used, the correct size product was generatedand this was the predominant product in each reaction.

The medium stringency annealing reaction comprised three differentannealing temperatures in each of the first three cycles. The annealingtemperature for the first cycle was 40° C. for 30 seconds; the annealingtemperature for the second cycle was 60° C. for 30 seconds; and theannealing temperature for the third cycle was 67° C. for 30 seconds.Annealing was performed at 67° C. for subsequent cycles untilcompletion. Similar to what was observed under low stringency annealingconditions, amplification of SNP TSC0087315 (FIG. 3B, lane 4) generatedmultiple bands under conditions of medium stringency. Amplification ofthe other three SNPs (lanes 1-3) produced a single band. These resultsdemonstrate that variable annealing temperatures can be used to cleanlyamplify loci of interest from genomic DNA with a primer that has anannealing length of 13 bases.

The high stringency annealing reaction was comprised of three differentannealing temperatures in each of the first three cycles. The annealingtemperature of the first cycle was 46° C. for 30 seconds; the annealingtemperature of the second cycle was 65° C. for 30 seconds; and theannealing temperature for the third cycle was 72° C. for 30 seconds.Annealing was performed at 72° C. for subsequent cycles untilcompletion. As shown in the photograph of the gel (FIG. 3C),amplification of SNP TSC0087315 (lane 4) using the high stringencyannealing temperatures generated a single band of the correct molecularweight. By raising the annealing temperatures for each of the firstthree cycles, non-specific amplification was eliminated. Amplificationof SNP TSC0095512 (lane 2) generated a single band. SNPs HC21S00340(lane 1), and TSC0214366 (lane 3) failed to amplify at the highstringency annealing temperatures, however, at the medium stringencyannealing temperatures, these SNPs amplified as a single band. Theseresults demonstrate that variable annealing temperatures can be used toreduce non-specific PCR products, as demonstrated for SNP TSC0087315(FIG. 3, lane 4).

Example 2

SNPs on chromosomes 1 (TSC0095512), 13 (TSC0264580), and 21 (HC21S00027)were analyzed. SNP TSC0095512 was analyzed using two different sets ofprimers, and SNP HC21S00027 was analyzed using two types of reactionsfor the incorporation of nucleotides.

Preparation of Template DNA

The template DNA was prepared from a 5 ml sample of blood obtained byvenipuncture from a human volunteer with informed consent. Template DNAwas isolated using the QIAmp DNA Blood Midi Kit supplied by QIAGEN(Catalog number 51183). The template DNA was isolated as perinstructions included in the kit. Following isolation, template DNA fromthirty-six human volunteers were pooled together and cut with therestriction enzyme EcoRI. The restriction enzyme digestion was performedas per manufacturer's instructions.

Primer Design

SNP HC21S00027 was amplified by PCR using the following primer set:

First primer: (SEQ ID NO: 17) 5′ ATAACCGTATGCGAATTCTATAATTTTCCTGATAAAGG3′ Second primer: (SEQ ID NO: 18) 5′ CTTAAATCAGGGGACTAGGTAAACTTCA 3′.

The first primer contained a biotin tag at the extreme 5′ end, and thenucleotide sequence for the restriction enzyme EcoRI. The second primercontained the nucleotide sequence for the restriction enzyme BsmF I(FIG. 4A).

Also, SNP HC21S00027 was amplified by PCR using the same first primerbut a different second primer with the following sequence:

Second primer: 5′ CTTAAATCAGACGGCTAGGTAAACTTCA 3′ (SEQ ID NO: 19)

This second primer contained the recognition site for the restrictionenzyme BceA I (FIG. 4B).

SNP TSC0095512 was amplified by PCR using the following primers:

First primer: (SEQ ID NO: 11) 5′ AAGTTTAGATCAGAATTCGTGAAAGCAGAAGTTGTCTG3′ Second primer: (SEQ ID NO: 20) 5′ TCTCCAACTAGGGACTCATCGAGTAAAG 3′.

The first primer had a biotin tag at the 5′ end and contained arestriction enzyme recognition site for EcoRI. The second primercontained a restriction enzyme recognition site for BsmF I (FIG. 4C).

Also, SNP TSC0095512 was amplified using the same first primer and adifferent second primer with the following sequence:

Second primer: 5′TCTCCAACTAACGGCTCATCGAGTAAAG 3′ (SEQ ID NO: 12)

This second primer contained the recognition site for the restrictionenzyme BceA I (FIG. 4D).

SNP TSC0264580, which is located on chromosome 13, was amplified withthe following primers:

First primer: (SEQ ID NO: 21) 5′ AACGCCGGGCGAGAATTCAGTTTTTCAACTTGCAAGG3′ Second primer: (SEQ ID NO: 22) 5′ CTACACATATCTGGGACGTTGGCCATCC 3′.

The first primer contained a biotin tag at the extreme 5′ end and had arestriction enzyme recognition site for EcoRI. The second primercontained a restriction enzyme recognition site for BsmF I.

PCR Reaction

All loci of interest were amplified from the template genomic DNA usingthe polymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and4,683,202, incorporated herein by reference). In this example, the lociof interest were amplified in separate reaction tubes but they couldalso be amplified together in a single PCR reaction. For increasedspecificity, a “hot-start” PCR was used. PCR reactions were performedusing the HotStarTaq Master Mix Kit supplied by QIAGEN (catalog number203443). The amount of template DNA and primer per reaction can beoptimized for each locus of interest but in this example, 40 ng oftemplate human genomic DNA and 5 μM of each primer were used. Fortycycles of PCR were performed. The following PCR conditions were used:

(1) 95° C. for 15 minutes and 15 seconds;

(2) 37° C. for 30 seconds;

(3) 95° C. for 30 seconds;

(4) 57° C. for 30 seconds;

(5) 95° C. for 30 seconds;

(6) 64° C. for 30 seconds;

(7) 95° C. for 30 seconds;

(8) Repeat steps 6 and 7 thirty nine (39) times;

(9) 72° C. for 5 minutes.

In the first cycle of PCR, the annealing temperature was about theinciting temperature of the 3′ annealing region of the second primers,which was 37° C. The annealing temperature in the second cycle of PCRwas about the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the first primer, which was 57° C. The annealingtemperature in the third cycle of PCR was about the melting temperatureof the entire sequence of the second primer, which was 64° C. Theannealing temperature for the remaining cycles was 64° C. Escalating theannealing temperature from TM1 to TM2 to TM3 in the first three cyclesof PCR greatly improves specificity. These annealing temperatures arerepresentative, and the skilled artisan will understand the annealingtemperatures for each cycle are dependent on the specific primers used.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results. The PCR products for SNP HC21S00027 and SNPTSC095512 are shown in FIGS. 5A-5D.

Purification of Fragment of Interest

The PCR products were separated from the genomic template DNA. Each PCRproduct was divided into four separate reaction wells of a Streptawell,transparent, High-Bind plate from Roche Diagnostics GmbH (catalog number1 645 692, as listed in Roche Molecular Biochemicals, 2001 BiochemicalsCatalog). The first primers contained a 5′ biotin tag so the PCRproducts bound to the Streptavidin coated wells while the genomictemplate DNA did not. The streptavidin binding reaction was performedusing a Thermomixer (Eppendorf) at 1000 rpm for 20 min. at 37° C. Eachwell was aspirated to remove unbound material, and washed three timeswith 1×PBS, with gentle mixing (Kandpal et al., Nucl. Acids Res.18:1789-1795 (1990); Kaneoka et al., Biotechniques 10:30-34 (1991);Green et al., Nucl. Acids Res. 18:6163-6164 (1990)).

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme thatbound the recognition site incorporated into the PCR products from thesecond primer. SNP HC21 S00027 (FIGS. 6A and 68) and SNP TSC0095512(FIGS. 6C and 6D) were amplified in separate reactions using twodifferent second primers. FIG. 6A (SNP HC21S00027) and FIG. 6C (SNPTSC0095512) depict the PCR products after digestion with the restrictionenzyme BsmF I (New England Biolabs catalog number R0572S). FIG. 6B (SNPHC21S00027) and FIG. 6D (SNP TSC0095512) depict the PCR products afterdigestion with the restriction enzyme BceA I (New England Biolabs,catalog number R0623 S). The digests were performed in the Streptawellsfollowing the instructions supplied with the restriction enzyme. SNPTSC0264580 was digested with BsmF I. After digestion with theappropriate restriction enzyme, the wells were washed three times withPBS to remove the cleaved fragments.

Incorporation of Labeled Nucleotide

The restriction enzyme digest described above yielded a DNA fragmentwith a 5′ overhang, which contained the SNP site or locus of interestand a 3′ recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide or nucleotides in the presence of a DNApolymerase.

For each SNP, four separate fill in reactions were performed; each ofthe four reactions contained a different fluorescently labeleddideoxynucleotide (ddATP, ddCTP, ddGTP, or ddTTP). The followingcomponents were added to each fill in reaction: 1 μl of a fluorescentlylabeled dideoxynucleotide, 0.5 μl of unlabeled ddNTPs (40 μM), whichcontained all nucleotides except the nucleotide that was fluorescentlylabeled, 2 μl of 10× sequenase buffer, 0.25 μl of Sequenase, and wateras needed for a 20 μl reaction. All of the fill in reactions wereperformed at 40° C. for 10 min. Non-fluorescently labeled nucleotideswas purchased from Fermentas Inc. (Hanover, Md.). All other labelingreagents were obtained from Amersham (Thermo Sequenase Dye TerminatorCycle Sequencing Core Kit, US 79565). In the presence of fluorescentlylabeled ddNTPs, the 3′ recessed end was extended by one base, whichcorresponds to the SNP or locus of interest (FIG. 7A-7D).

A mixture of labeled ddNTPs and unlabeled dNTPs also was used for the“fill in” reaction for SNP HC21S00027. The “fill in” conditions were asdescribed above except that a mixture containing 40 μM unlabeled dNTPs,1 μl fluorescently labeled ddATP, I fluorescently labeled ddCTP, 1 μlfluorescently labeled ddGTP, and 1 μl ddTTP was used. The fluorescentddNTPs were obtained from Amersham (Thermo Sequenase Dye TerminatorCycle Sequencing Core Kit, US 79565; Amersham did not publish theconcentrations of the fluorescent nucleotides). SNP HC21S00027 wasdigested with the restriction enzyme BsmF I, which generated a 5′overhang of four bases. As shown in FIG. 7E, if the first nucleotideincorporated is a labeled dideoxynucleotide, the 3′ recessed end isfilled in by one base, allowing detection of the SNP or locus ofinterest. However, if the first nucleotide incorporated is a dNTP, thepolymerase continues to incorporate nucleotides until a ddNTP is filledin. For example, the first two nucleotides can be filled in with dNTPs,and the third nucleotide with a ddNTP, allowing detection of the thirdnucleotide in the overhang. Thus, the sequence of the entire 5′ overhangcan be determined, which increases the information obtained from eachSNP or locus of interest.

After labeling, each Streptawell was rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments were then released from theStreptawells by digestion with the restriction enzyme EcoRI, accordingto the manufacturer's instructions that were supplied with the enzyme(FIGS. 8A-8D). Digestion was performed for 1 hour at 37° C. with shakingat 120 rpm.

Detection of the Locus of Interest

After release from the streptavidin matrix, 2-3 μl of the 10 μl samplewas loaded in a 48 well membrane tray (The Gel Company, catalog numberTAM48-01). The sample in the tray was absorbed with a 48 Flow MembraneComb (The Gel Company, catalog number AM48), and inserted into a 36 cm5% acrylamide (urea) gel (BioWhittaker Molecular Applications, LongRanger Run Gel Packs, catalog number 50691).

The sample was electrophoresed into the gel at 3000 volts for 3 min. Themembrane comb was removed, and the gel was run for 3 hours on an ABI 377Automated Sequencing Machine. The incorporated labeled nucleotide wasdetected by fluorescence.

As shown in FIG. 9A, from a sample of thirty six (36) individuals, oneof two nucleotides, either adenosine or guanine, was detected at SNPHC21S00027. These are the two nucleotides reported to exist at SNPHC21S00027 (http://snp.cshl.org/snpsearch.shtml).

One of two nucleotides, either guanine or cytosine, was detected at SNPTS00095512 (FIG. 9B). The same results were obtained whether the locusof interest was amplified with a second primer that contained arecognition site for BceA I or the second primer contained a recognitionsite for BsmF I.

As shown in FIG. 9C, one of two nucleotides was detected at SNPTSC0264580, which was either adenosine or cytosine. These are the twonucleotides reported for this SNP site(http://snp.cshl.org/snpsearch.shtml). In addition, a thymidine wasdetected one base from the locus of interest. In a sequence dependentmanner, BsmF I cuts some DNA molecules at the 10/14 position and otherDNA molecules, which have the same sequence, at the 11/15 position. Whenthe restriction enzyme BsmF I cuts 11 nucleotides away on the sensestrand and 15 nucleotides away on the antisense strand, the 3′ recessedend is one base from the SNP site. The sequence of SNP TSC0264580indicated that the base immediately preceding the SNP site was athymidine. The incorporation of a labeled ddNTP into this positiongenerated a fragment one base smaller than the fragment that was cut atthe 10/14 position. Thus, the DNA molecules cut at the 11/15 positionprovided sequence information about the base immediately preceding theSNP site, and the DNA molecules cut at the 10/14 position providedsequence information about the SNP site.

SNP HC21S00027 was amplified using a second primer that contained therecognition site for BsmF I. A mixture of labeled ddNTPs and unlabeleddNTPs was used to fill in the 5′ overhang generated by digestion withBsmF I. If a dNTP was incorporated, the polymerase continued toincorporate nucleotides until a ddNTP was incorporated. A population ofDNA fragments, each differing by one base, was generated, which allowedthe full sequence of the overhang to be determined.

As seen in FIG. 9D, an adenosine was detected, which was complementaryto the nucleotide (a thymidine) immediately preceding the SNP or locusof interest. This nucleotide was detected because of the 11/15 cuttingproperty of BsmF I, which is described in detail above. A guanine and anadenosine were detected at the SNP site, which are the two nucleotidesreported for this SNP site (FIG. 9A). The two nucleotides were detectedat the SNP site because the molecular weights of the dyes differ, whichallowed separation of the two nucleotides. The next nucleotide detectedwas a thymidine, which is complementary to the nucleotide immediatelydownstream of the SNP site. The next nucleotide detected was a guanine,which was complementary to the nucleotide two bases downstream of theSNP site. Finally, an adenosine was detected, which was complementary tothe third nucleotide downstream of the SNP site. Sequence informationwas obtained not only for the SNP site but for the nucleotideimmediately preceding the SNP site and the next three nucleotides.

None of the loci of interest contained a mutation. However, if one ofthe loci of interest harbored a mutation including but not limited to apoint mutation, insertion, deletion, translocation or any combination ofsaid mutations, it could be identified by comparison to the consensus orpublished sequence. Comparison of the sequences attributed to each ofthe loci of interest to the native, non-disease related sequence of thegene at each locus of interest determines the presence or absence of amutation in that sequence. The finding of a mutation in the sequence isthen interpreted as the presence of the indicated disease, or apredisposition to develop the same, as appropriate, in that individual.The relative amounts of the mutated vs. normal or non-mutated sequencecan be assessed to determine if the subject has one or two alleles ofthe mutated sequence, and thus whether the subject is a carrier, orwhether the indicated mutation results in a dominant or recessivecondition.

Example 3

Four loci of interest from chromosome 1 and two loci of interest fromchromosome 21 were amplified in separate PCR reactions, pooled together,and analyzed. The primers were designed so that each amplified locus ofinterest was a different size, which allowed detection of the loci ofinterest.

Preparation of Template DNA

The template DNA was prepared from a 5 ml sample of blood obtained byvenipuncture from a human volunteer with informed consent. Template DNAwas isolated using the QIAmp DNA Blood Midi Kit supplied by QIAGEN(Catalog number 51183). The template DNA was isolated as perinstructions included in the kit. Template DNA was isolated fromthirty-six human volunteers, and then pooled into a single sample forfurther analysis.

Primer Design

SNP TSC 0087315 was amplified using the following primers:

First primer: (SEQ ID NO: 15) 5′TTACAATGCATGAATTCATCTTGGTCTCTCAAAGTGC 3′Second primer: (SEQ ID NO: 16) 5′TGGACCATAAACGGCCAAAAACTGTAAG3′.

SNP TSC0214366 was amplified using the following primers:

First primer: (SEQ ID NO: 13) 5′ATGACTAGCTATGAATTCGTTCAAGGTAGAAAATGGAA3′ Second primer: (SEQ ID NO: 14) 5′GAGAATTAGAACGGCCCAAATCCCACTC 3′

SNP TSC 0413944 was amplified with the following primers:

First primer: (SEQ ID NO: 23) 5′ TACCTTTTGATCGAATTCAAGGCCAAAAATATTAAGTT3′ Second primer: (SEQ ID NO: 24) 5′ TCGAACTTTAACGGCCTTAGAGTAGAGA 3′

SNP TSC0095512 was amplified using the following primers;

First primer: (SEQ ID NO: 11) 5′AAGTTTAGATCAGAATTCGTGAAAGCAGAAGTTGTCTG3′ Second primer: (SEQ ID NO: 12) 5′TCTCCAACTAACGGCTCATCGAGTAAAG 3′

SNP HC21S00131 was amplified with the following primers:

First primer: (SEQ ID NO: 25) 5′ CGATTTCGATAAGAATTCAAAAGCAGTTCTTAGTTCAG3′ Second primer: (SEQ ID NO: 26) 5′TGCGAATCTTACGGCTGCATCACATTCA 3′

SNP HC21S00027 was amplified with the following primers:

First primer: (SEQ ID NO: 17) 5′ ATAACCGTATGCGAATTCTATAATTTTCCTGATAAAGG3′ Second primer: (SEQ ID NO: 19) 5′ CTTAAATCAGACGGCTAGGTAAACTTCA 3′

For each SNP, the first primer contained a recognition site for therestriction enzyme EcoRI and had a biotin tag at the extreme 5′ end. Thesecond primer used to amplify each SNP contained a recognition site forthe restriction enzyme BceA I.

PCR Reaction

The PCR reactions were performed as described in Example 2 except thatthe following annealing temperatures were used: the annealingtemperature for the first cycle of PCR was 37° C. for 30 seconds, theannealing temperature for the second cycle of PCR was 57° C. for 30seconds, and the annealing temperature for the third cycle of PCR was64° C. for 30 seconds. All subsequent cycles had an annealingtemperature of 64° C. for 30 seconds. Thirty seven (37) cycles of PCRwere performed. After PCR, ¼ of the volume was removed from eachreaction, and combined into a single tube.

Purification of Fragment of Interest

The PCR products (now combined into one sample, and referred to as “thesample”) were separated from the genomic template DNA as described inExample 2 except that the sample was bound to a single well of aStreptawell microtiter plate.

Restriction Enzyme Digestion of Isolated Fragments

The sample was digested with the restriction enzyme BceA I, which boundthe recognition site in the second primer. The restriction enzymedigestions were performed following the instructions supplied with theenzyme. After the restriction enzyme digest, the wells were washed threetimes with 1×PBS.

Incorporation of Nucleotides

The restriction enzyme digest described above yielded DNA molecules witha 5′ overhang, which contained the SNP site or locus of interest and a3′ recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide in the presence of a DNA polymerase.

The following components were used for the fill in reaction: 1 μl offluorescently labeled ddATP; 1 μl of fluorescently labeled ddTTP; 1 μlof fluorescently labeled ddGTP; 1 μl of fluorescently labeled ddCTP; 2μl of 10× sequenase buffer, 0.25 μl of Sequenase, and water as neededfor a 20 μl reaction. The fill in reaction was performed at 40° C. for10 min. All labeling reagents were obtained from Amersham (ThermoSequenase Dye Terminator Cycle Sequencing Core Kit (US 79565); theconcentration of the ddNTPS provided in the kit is proprietary and notpublished by Amersham). In the presence of fluorescently labeled ddNTPs,the 3′ recessed end was filled in by one base, which corresponds to theSNP or locus of interest.

After the incorporation of nucleotide, the Streptawell was rinsed with1×PBS (100 μl) three times. The “filled in” DNA fragments were thenreleased from the Streptawell by digestion with the restriction enzymeEcoRI following the manufacturer's instructions. Digestion was performedfor 1 hour at 37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

After release from the streptavidin matrix, 2-3 μl of the 10 μl samplewas loaded in a 48 well membrane tray (The Gel Company, catalog numberTAM48-01). The sample in the tray was absorbed with a 48 Flow MembraneComb (The Gel Company, catalog number AM48), and inserted into a 36 cm5% acrylamide (urea) gel (BioWhittaker Molecular Applications, LongRanger Run Gel Packs, catalog number 50691).

The sample was electrophoresed into the gel at 3000 volts for 3 min. Themembrane comb was removed, and the gel was run for 3 hours on an ABI 377Automated Sequencing Machine. The incorporated nucleotide was detectedby fluorescence.

The primers were designed so that each amplified locus of interestdiffered in size. As shown in FIG. 10, each amplified loci of interestdiffered by about 5-10 nucleotides, which allowed the loci of interestto be separated from one another by gel electrophoresis. Two nucleotideswere detected for SNP TSC0087315, which were guanine and cytosine. Theseare the two nucleotides reported to exist at SNP TSC0087315(http://snp.cshl.org/snpsearch.shtml). The sample comprised template DNAfrom 36 individuals and because the DNA molecules that incorporated aguanine differed in molecular weight from those that incorporated acytosine, distinct bands were seen for each nucleotide.

Two nucleotides were detected at SNP HC21S00027, which were guanine andadenosine (FIG. 10). The two nucleotides reported for this SNP site areguanine and adenosine (http://snp.cshl.org/snpsearch.shtml). Asdiscussed above, the sample contained template DNA from thirty-sixindividuals, and one would expect both nucleotides to be represented inthe sample. The molecular weight of the DNA fragments that incorporateda guanine was distinct from the DNA fragments that incorporated anadenosine, which allowed both nucleotides to be detected.

The nucleotide cytosine was detected at SNP TSC0214366 (FIG. 10). Thetwo nucleotides reported to exist at this SNP position are thymidine andcytosine.

The nucleotide guanine was detected at SNP TSC0413944 (FIG. 10). The twonucleotides reported for this SNP are guanine and cytosine(http://spp.cshl.org/snpsearch.shtml).

The nucleotide cytosine was detected at SNP TS00095512 (FIG. 10). Thetwo nucleotides reported for this SNP site are guanine and cytosine(http://snp.cshl.org/snpsearch.shtml).

The nucleotide detected at SNP HC21S00131 was guanine. The twonucleotides reported for this SNP site are guanine and adenosine(http://snp.cshl.org/snpsearch.shtml).

As discussed above, the sample was comprised of DNA templates fromthirty-six individuals and one would expect both nucleotides at the SNPsites to be represented. For SNP TSC0413944, TSC0095512, TSC0214366 andHC21S00131, one of the two nucleotides was detected. It is likely thatboth nucleotides reported for these SNP sites are present in the samplebut that one fluorescent dye overwhelms the other. The molecular weightof the DNA molecules that incorporated one nucleotide did not allowefficient separation of the DNA molecules that incorporated the othernucleotide. However, the SNPs were readily separated from one another,and for each SNP, a proper nucleotide was incorporated. The sequences ofmultiple loci of interest from multiple chromosomes, which were treatedas a single sample after PCR, were determined.

A single reaction containing fluorescently labeled ddNTPs was performedwith the sample that contained multiple loci of interest. Alternatively,four separate fill in reactions can be performed where each reactioncontains one fluorescently labeled nucleotide (ddATP, ddTTP, ddGTP, orddCTP) and unlabeled ddNTPs (see Example 2, FIGS. 7A-7D and FIGS. 9A-C).Four separate “fill in” reactions will allow detection of any nucleotidethat is present at the loci of interest. For example, if analyzing asample that contains multiple loci of interest from a single individual,and said individual is heterozygous at one or more than one loci ofinterest, four separate “fill in” reactions can be used to determine thenucleotides at the heterozygous loci of interest.

Also, when analyzing a sample that contains templates from multipleindividuals, four separate “fill in” reactions will allow detection ofnucleotides present in the sample, independent of how frequent thenucleotide is found at the locus of interest. For example, if a samplecontains DNA templates from 50 individuals, and 49 of the individualshave a thymidine at the locus of interest, and one individual has aguanine, the performance of four separate “fill in” reactions, whereineach “fill in” reaction is run in a separate lane of a gel, such as inFIGS. 9A-9C, will allow detection of the guanine. When analyzing asample comprised of multiple DNA templates, multiple “fill in” reactionswill alleviate the need to distinguish multiple nucleotides at a singlesite of interest by differences in mass.

In this example, multiple single nucleotide polymorphisms were analyzed.It is also possible to determine the presence or absence of mutations,including but not limited to point mutations, transitions,transversions, translocations, insertions, and deletions from multipleloci of interest. The multiple loci of interest can be from a singlechromosome or from multiple chromosomes. The multiple loci of interestcan be from a single gene or from multiple genes.

The sequence of multiple loci of interest that cause or predispose to adisease phenotype can be determined. For example, one could amplify oneto tens to hundreds to thousands of genes implicated in cancer or anyother disease. The primers can be designed so that each amplified lociof interest differs in size. After PCR, the amplified loci of interestcan be combined and treated as a single sample. Alternatively, themultiple loci of interest can be amplified in one PCR reaction or thetotal number of loci of interest, for example 100, can be divided intosamples, for example 10 loci of interest per PCR reaction, and thenlater pooled. As demonstrated herein, the sequence of multiple loci ofinterest can be determined. Thus, in one reaction, the sequence of oneto ten to hundreds to thousands of genes that predispose or cause adisease phenotype can be determined.

Example 4

The ability to determine the sequence or detect chromosomalabnormalities of a fetus using free fetal DNA in a sample from apregnant female has been hindered by the low percentage of free fetalDNA. Increasing the percentage of free fetal DNA would enhance thedetection of mutation, insertion, deletion, translocation, transversion,monosomy, trisomy, trisomy 21, trisomy 18, trisomy 13, XXY, XXX, otheraneuploidies, deletion, addition, amplification, translocation andrearrangement. The percent of fetal DNA in plasma obtained from apregnant female was determined both in the absence and presence ofinhibitors of cell lysis. A genetic marker on the Y chromosome was usedto calculate the percent of fetal DNA.

Preparation of Template DNA

The DNA template was prepared from a 5 ml sample of blood obtained byvenipuncture from a human volunteer with informed consent. The blood wasaliquoted into two tubes (Fischer Scientific, 9 ml EDTA Vacuette tubes,catalog number NC9897284). Formaldehyde (25 μl/ml of blood) was added toone of the tubes. The sample in the other tube remained untreated,except for the presence of the EDTA. The tubes were spun at 1000 rpm forten minutes. Two milliliters of the supernatant (the plasma) of eachsample was transferred to a new tube and spun at 3000 rpm for tenminutes. 800 μl of each sample was used for DNA purification. DNA wasisolated using the Qiagen Midi Kit for purification of DNA from bloodcells (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA was elutedin 100 μl of distilled water. Two DNA templates were obtained: one fromthe blood sample treated with EDTA, and one from the blood sampletreated with EDTA and formaldehyde.

Primer Design

Two different sets of primers were used: one primer set was specific forthe Y chromosome, and thus specific for fetal DNA, and the other primerset was designed to amplify the cystic fibrosis gene, which is presenton both maternal template DNA and fetal template DNA.

In this example, the first and second primers were designed so that theentire 5′ and 3′ sequence of each primer annealed to the template DNA.In this example, the fetus had an XY genotype, and the Y chromosome wasused as a marker for the presence of fetal DNA. The following primerswere designed to amplify the SRY gene on the Y chromosome.

First primer: (SEQ ID NO: 263) 5′ TGGCGATTAAGTCAAATTCGC 3′ Secondprimer: (SEQ ID NO: 264) 5 CCCCCTAGTACCCTGACAATGTATT 3′

Primers designed to amplify any gene, or region of a region, or any partof any chromosome could be used to detect maternal and fetal DNA. Inthis example, the following primers were designed to amplify the cysticfibrosis gene:

First primer: (SEQ ID NO: 265) 5′ CTGTTCTGTGATATTATGTGTGGT 3′ Secondprimer: (SEQ ID NO: 266) 5′ AATTGTTGGCATTCCAGCATTG 3′

PCR Reaction

The SRY gene and the cystic fibrosis gene were amplified from thetemplate genomic DNA using PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202).For increased specificity, a “hot-start” PCR was used. PCR reactionswere performed using the HotStarTaq Master Mix Kit supplied by Qiagen(Catalog No. 203443). For amplification of the SRY gene, the DNA elutedfrom the Qiagen purification column was diluted serially 1:2. Foramplification of the cystic fibrosis gene, the DNA from the Qiagenpurification column was diluted 1:4, and then serially diluted 1:2. Thefollowing components were used for each PCR reaction: 8 μl of templateDNA (diluted or undiluted), 1 μl of each primer (5 μM), 10 μl of HotStarTag mix. The following PCR conditions were used:

(1) 950 C for 15′

(2) 94° C. for 1′

(3) 54° C. for 15″

(4) 72° C. for 30″

(5) Repeat steps 2-4 for 45 cycles.

(6) 10′ at 72° C.

Quantification of Fetal DNA

The DNA templates that were eluted from the Qiagen columns were seriallydiluted to the following concentrations: 1:2, 1:4, 1:8, 1:16, 1:32,1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048, and 1:4096. Amplification ofthe SRY gene was performed using the templates that were undiluted, 1:2,1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512. Amplification of thecystic fibrosis gene was performed using the DNA templates that werediluted 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048,and 1:4096. The same dilution series was performed with the DNAtemplates that were purified from the plasma sample treated with EDTAalone and the plasma sample treated with EDTA and formaldehyde.

The results of the PCR reactions using the DNA template that wasisolated from the plasma sample treated with EDTA are shown in FIG. 11A.The SRY gene was amplified from the undiluted DNA template, and also inthe sample that was diluted 1:2 (FIG. 11A). The SRY gene was notamplified in the next seven serial dilutions. On the other hand, thecystic fibrosis gene was detected in the serial dilutions up to 1:256. Agreater presence of the cystic fibrosis gene was expected because of thehigher percentage of maternal DNA present in the plasma. The lastdilution sample that provided for amplification of the gene product wasassumed to have one copy of the cystic fibrosis gene or the SRY gene.

The results of the PCR reactions using the DNA template that wasisolated from the plasma sample treated with formaldehyde and EDTA areshown in FIG. 11B. The SRY gene was amplified from the undiluted DNAtemplate, and also in the sample that was diluted 1:2 (FIG. 11B). TheSRY gene was not amplified in the next six dilutions. However, in the1:256 dilution, the SRY gene was detected. It is unlikely that theamplification in the 1:256 sample represents a real signal because theprior six dilution series were all negative for amplification of SRY.Amplification of the SRY gene in this sample was likely an experimentalartifact resulting from the high number of PCR cycles used. Thus, the1:256 sample was not used in calculating the amount of fetal DNA presentin the sample.

Amplification of the cystic fibrosis gene was detected in the samplethat was diluted 1:16 (FIG. 11B). The presence of the formal in preventsmaternal cell lysis, and thus, there is a lower percentage of maternalDNA in the sample. This is in strong contrast to the sample that wastreated with only EDTA, which supported amplification up to a dilutionof 1:256.

The percent of fetal DNA present in the maternal plasma was calculatedusing the following formula:

% fetal DNA=(amount of SRY gene/amount of cystic fibrosis gene)*2*100.

The amount of SRY gene was represented by the highest dilution value inwhich the gene was amplified. Likewise, the amount of cystic fibrosisgene was represented by the highest dilution value in which it wasamplified. The formula contains a multiplication factor of two (2),which is used to normalize for the fact that there is only one copy ofthe SRY gene (located on the Y chromosome), while there are two copiesof the cystic fibrosis gene.

For the above example, the percentage of fetal DNA present in the samplethat was treated with only EDTA was 1.56% (2/256*2*100). The reportedpercentage of fetal DNA present in the plasma is between 0.39-11.9%(Pertl and Bianchi, Obstetrics and Gynecology, Vol, 98, No. 3, 483-490(2001). The percentage of fetal DNA present in the sample treated withformalin and EDTA was 25% (2/16*2*100). The experiment was repeatednumerous times, and each time the presence of formalin increased theoverall percentage of fetal DNA.

The percent fetal DNA from eighteen blood samples with and withoutformalin was calculated as described above with the exception thatserial dilutions of 1:5 were performed. As 1:5 dilutions were performed,the last serial dilution that allowed detection of either the SRY geneor the cystic fibrosis gene may have had one copy of the gene or it mayhave had 4 copies of the gene. The results from the eighteen sampleswith and without formalin are summarized in Table V. The low rangeassumes that the last dilution sample had one copy of the genes and thehigh range assumes that the last dilution had four copies of the genes.

TABLE V Mean Percentage Fetal DNA with and without formalin. SampleLower Range Upper Range Formalin 19.47 43.69 Without Formalin 7.71 22.1

An overall increase in fetal DNA was achieved by reducing the maternalcell lysis, and thus, reducing the amount of maternal DNA present in thesample. In this example, formaldehyde was used to prevent lysis of thecells, however any agent that prevents the lysis of cells or increasesthe structural integrity of the cells can be used. Two or more than twocell lysis inhibitors can be used. The increase in fetal DNA in thematernal plasma allows the sequence of the fetal DNA to be determined,and provides for the rapid detection of abnormal DNA sequences orchromosomal abnormalities including but not limited to point mutation,reading frame shift, transition, transversion, addition, insertion,deletion, addition-deletion, frame-shift, missense, reverse mutation,and microsatellite alteration, trisomy, monosomy, other aneuploidies,amplification, rearrangement, translocation, transversion, deletion,addition, amplification, fragment, translocation, and rearrangement.

Example 5

A DNA template from an individual with a genotype of trisomy 21 wasanalyzed. Three loci of interest were analyzed on chromosome 13 and twoloci of interest were analyzed on chromosome 21.

Preparation of Template DNA

The template DNA was prepared from a 5 ml sample of blood obtained byvenipuncture from a human volunteer with informed consent. The humanvolunteer had previously been genotyped to have an additional chromosome21 (trisomy 21). Template DNA was isolated using QIAamp DNA Blood MidiKit supplied by QIAGEN (Catalog number 51183).

Primer Design

The following five single nucleotide polymorphisms were analyzed: SNPTSC 0115603 located on chromosome 21; SNP TSC 03209610 located onchromosome 21; SNP TSC 0198557 located on chromosome 13; and SNP TSC0200347 located on chromosome 13. The DNA template from anotherindividual was used as an internal control. The SNP TSC 0200347, whichwas previously identified as being homozygous for guanine, was used asthe internal control. The SNP Consortium Ltd database can be accessed athttp://snp.cshl.org/, website address effective as of Apr. 1, 2002.

SNP TSC 0115603 was amplified using the following primers:

First Primer: (SEQ ID NO: 267) 5′ GTGCACTTACGTGAATTCAGATGAACGTGATGTAGTAG3′ Second Primer: (SEQ ID NO: 268) 5′ TCCTCGTACTCAACGGCTTTCTCTGAAT 3′

The first primer was biotinylated at the 5′ end, and contained therestriction enzyme recognition site for EcoR I. The second primercontained the restriction enzyme recognition site for the restrictionenzyme BceA I.

SNP TSC 0309610 was amplified using the following primers:

First primer: (SEQ ID NO: 269) 5′ TCCGGAACACTAGAATTCTTATTTACATACACACTTGT3′ Second primer: (SEQ ID NO: 270) 5′ CGAATAAGGTAGACGGCAACAATGAGAA 3′

The first primer contained a biotin group at the 5′ end, and arestriction enzyme recognition site for the restriction enzyme EcoR I.The second primer contained the restriction enzyme recognition site forBceA I.

Submitted SNP (ss) 813773 (accession number assigned by the NCBISubmitted SNP (ss) Database) was amplified with the following primers:

First primer: (SEQ ID NO: 271) 5′ CGGTAAATCGGAGAATTCAGAGGATTTAGAGGAGCTAA3′ Second primer: (SEQ ID NO: 272) 5′ CTCACGTTCGTTACGGCCATTGTGATAGC 3′

The first primer contains a biotin group at the 5′ end, and arecognition site for the restriction enzyme EcoR I. The second primercontained the restriction enzyme recognition site for BceA I.

SNP TSC 0198557 was amplified with the following primers:

First primer: (SEQ ID NO: 273) 5′ GGGGAAACAGTAGAATTCCATATGGACAGAGCTGTACT3′ Second primer: (SEQ ID NO: 274) 5′ TGAAGCTGTCGGACGGCCTTTGCCCTCTC 3′

The first primer contains a biotin group at the 5′ end, and arecognition site for the restriction enzyme EcoR I. The second primercontained the restriction enzyme recognition site for BceA I.

SNP TSC 0197279 was amplified with the following primers:

First primer: (SEQ ID NO: 275) 5′ ATGGGCAGTTATGAATTCACTACTCCCTGTAGCTTGTT3′ Second primer: (SEQ ID NO: 276) 5′ TGATTGGCGCGAACGGCACTCAGAGAAGA 3′

The first primer contained a biotin group at the 5′ end, and arecognition site for the restriction enzyme for EcoR I. The secondprimer contained the recognition site for the restriction enzyme BceA I.

SNP TSC 0200347 was amplified with the following primers:

First primer: (SEQ ID NO: 277) 5′ CTCAAGGGGACCGAATTCGCTGGGGTCTTCTGTGGGTC3′ Second primer: (SEQ ID NO: 278) 5′ TAGGGCGGCGTGACGGCCAGCCAGTGGT 3′

The first primer contained a biotin group at the 5′ end, and therecognition site for the restriction enzyme EcoR I. The second primercontained the restriction enzyme recognition site for BceA I.

PCR Reaction

All five loci of interest were amplified from the template genomic DNAusing PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202). For increasedspecificity, a “hot-start” PCR was used. PCR reactions were performedusing the HotStarTaq Master Mix Kit supplied by QIAGEN (catalog number203443). The amount of template DNA and primer per reaction can beoptimized for each locus of interest; in this example, 40 ng of templatehuman genomic DNA and 5 μM of each primer were used. Thirty-eight cyclesof PCR were performed. The following PCR conditions were used for SNPTSC 0115603, SNP TSC 0309610, and SNP TSC 02003437:

(1) 95° C. for 15 minutes and 15 seconds;

(2) 42° C. for 30 seconds;

(3) 95° C. for 30 seconds;

(4) 60° C. for 30 seconds;

(5) 95° C. for 30 seconds;

(6) 69° C. for 30 seconds;

(7) 95° C. for 30 seconds;

(8) Repeat steps 6 and 7 thirty nine (37) times;

(9) 72° C. for 5 minutes.

The following PCR conditions were used for SNP ss813773, SNP TSC0198557, and SNP TSC 0197279:

(1) 95° C. for 15 minutes and 15 seconds;

(2) 37° C. for 30 seconds;

(3) 95° C. for 30 seconds;

(4) 57° C. for 30 seconds;

(5) 95° C. for 30 seconds;

(6) 64° C. for 30 seconds;

(7) 95° C. for 30 seconds;

(8) Repeat steps 6 and 7 thirty nine (37) times; and

(9) 72° C. for 5 minutes.

In the first cycle of each PCR, the annealing temperature was about themelting temperature of the 3′ annealing region of the second primer. Theannealing temperature in the second cycle of PCR was about the meltingtemperature of the 3′ region, which anneals to the template DNA, of thefirst primer. The annealing temperature in the third cycle of PCR wasabout the melting temperature of the entire sequence of the secondprimer. Escalating the annealing temperature from TM1 to TM2 to TM3 inthe first three cycles of PCR greatly improves specificity. Theseannealing temperatures are representative, and the skilled artisan willunderstand the annealing temperatures for each cycle are dependent onthe specific primers used. The temperatures and times for denaturing,annealing, and extension, can be optimized by trying various settingsand using the parameters that yield the best results.

Purification of Fragment of Interest

PCR products were separated from the components of the PCR reactionusing Qiagen's MinElute PCR Purification Kit following manufacturer'sinstructions (Catalog number 28006). The PCR products were eluted in 20μl of distilled water. For each amplified SNP, one microliter of PCRproduct, 1 μl of amplified internal control DNA (SNP TSC 0200347), and 8μl of distilled water were mixed. Five microliters of each sample wasplaced into two separate reaction wells of a Pierce StreptaWellMicrotiter plate (catalog number 15501). The first primers contained a5′ biotin tag so the PCR products bound to the Streptavidin coated wellswhile the genomic template DNA did not. The streptavidin bindingreaction was performed using a Thermomixer (Eppendorf) at 150 rpm for 1hour at 45° C. Each well was aspirated to remove unbound material, andwashed three times with 1×PBS, with gentle mixing (Kandpal et al., Nucl.Acids Res. 18:1789-1795 (1990); Kaneoka et al., Biotechniques 10:30-34(1991); Green et al., Nucl. Acids Res. 18:6163-6164 (1990)).

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme thatbound the recognition site that was incorporated into the PCR productsfrom the second primer. The purified PCR products were digested with therestriction enzyme BceA I (New England Biolabs, catalog number R0623S).The digests were performed in the wells of the microtiter platefollowing the instructions supplied with the restriction enzyme. Afterdigestion with the appropriate restriction enzyme, the wells were washedthree times with PBS to remove the cleaved fragments.

Incorporation of Labeled Nucleotide

The restriction enzyme digest described above yielded a DNA fragmentwith a 5′ overhang, which contained the SNP and a 3′ recessed end. The5′ overhang functioned as a template allowing incorporation of anucleotide or nucleotides in the presence of a DNA polymerase.

For each SNP, two fill in reactions were performed; each reactioncontained a different fluorescently labeled dideoxynucleotide (ddATP,ddCTP, ddGTP, or ddTTP, depending on the reported nucleotides to existat a particular SNP). For example, the nucleotides adenine and thymidinehave been reported at SNP TSC 0115603. Therefore, the digested PCRproduct for SNP TSC 0115603 was mixed with either fluorescently labeledddATP or fluorescently labeled ddTTP. Each reaction containedfluorescently labeled ddGTP for the internal control. The followingcomponents were added to each fill in reaction: 2 μl of a ROX-conjugateddideoxynucleotide (depending on the nucleotides reported for each SNP),2 μl of ROX-conjugated ddGTP (internal control), 2.5 μl of 10× sequenasebuffer, 2 μl of Sequenase, and water as needed for a 25 μl reaction. Allof the fill in reactions were performed at 45° C. for 45 min. However,shorter time periods of incorporation can be used. Non-fluorescentlylabeled ddNTPs were purchased from Fermentas Inc. (Hanover, Md.). TheROX-conjugated ddNTPs were obtained from Perkin Elmer. In the presenceof fluorescently labeled ddNTPs, the 3′ recessed end was extended by onebase, which corresponds to the SNP or locus of interest.

After labeling, each Streptawell was rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments were then released from theStreptawells by digestion with the restriction enzyme EcoR I followingmanufacturer's recommendations. Digestion was performed for 1 hour at37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

After release from the streptavidin matrix, 3 μl of the 10 μl sample wasloaded in a 48 well membrane tray (The Gel Company, catalog numberTAM48-01). The sample in the tray was absorbed with a 48 Flow MembraneComb (The Gel Company, catalog number AM48), and inserted into a 36 cm5% acrylamide (urea) gel (BioWhittaker Molecular Applications, LongRanger Run Gel Packs, catalog number 50691).

The sample was electrophoresed into the gel at 3000 volts for 3 min. Themembrane comb was removed, and the gel was run for 3 hours on an ABI 377Automated Sequencing Machine. The incorporated labeled nucleotide wasdetected by fluorescence.

As seen in FIG. 12, SNP TSC 0115603 was “filled in” with labeled ddTTP(lane 1) and in a separate reaction with labeled ddATP (lane 3). Thecalculated ratio between the nucleotides, using the raw data, was 66:34,which is consistent with the theoretical ratio of 66:33 for a SNP onchromosome 21 in an individual with trisomy 21. Both the ddTTP and ddATPwere labeled with the same fluorescent dye to minimize variability inincorporation efficiencies of the dyes. However, nucleotides withdifferent fluorescent labels or any detectable label can be used. It ispreferable to calculate the coefficients of incorporation when differentlabels are used.

Each fill in reaction was performed in a separate well so it waspossible that there could be variability in DNA binding between thewells of the microtiter plate. To account for the potential variabilityof DNA binding to the streptavidin-coated plates, an internal controlwas used. The internal control (SNP TSC 0200347), which is homozygousfor guanine, was added to the sample prior to splitting the sample intotwo separate wells, and thus, an equal amount of the internal controlshould be present in each well. The amount of incorporated ddGTP can befixed between the two reactions. If the amount of DNA in each well isequal, the amount of incorporated ddGTP should be equal because thereaction is performed under saturating conditions, with saturatingconditions being defined as conditions that support incorporation of anucleotide at each template molecule. Using the internal control, theratio of incorporated ddATP to ddTTP was 614:36.6, This ratio was verysimilar to the ratio obtained with the raw data, indicating that thereare minor differences in the two fill in reactions for a particular SNP.

TABLE VI Allele Frequencies at Multiple SNPs on DNA Template fromIndividual with Trisomy 21 Peak Allele Internal Allele SNP Allele AreaRatio Control Normalized Peak Area Ratio (%) TSC A 5599 66 723 5599 63.40115603 T 2951 34 661 3227 ((723/661)*2951) 36.6 TSC T 4126 64 1424 412666.8 0309610 C 2342 36 1631 2045 ((1424/1631)*2342) 33.2 ss813773 A 419946 808 4199 41 C 4870 54 647 6082 ((808/647)*4870) 59 TSC T 3385 55 7193385 49 0198557 C 2741 45 559 3525 719/559 *2741) 51 TSC T 8085 53 27528085 50.7 0197279 C 7202 47 2520 7865 (2752/2520 *7202 49.3

SNP TSC 0309610 was filled in with ddTTP (lane 3) or ddCTP (lane 4)(FIG. 12). The calculated ratio for the nucleotides, using the raw data,was 64:36. Both ddTTP and ddCTP were labeled with the same fluorescentdye. After normalization to the internal control, as discussed above,the calculated allele ratio of ddTTP to ddCTP was 66.8:33.2 (Table VI).Again, both the calculated ratio from the raw data and the calculatedratio using the internal control are very similar to the theoreticalratio of 66.6:33.4 for a SNP on chromosome 21 in an individual withtrisomy.

To demonstrate that the 66:33 ratios for nucleotides at heterozygousSNPS represented loci on chromosomes present in three copies, SNPs onchromosome 13 were analyzed. The individual from whom the blood samplewas obtained had previously been genotyped with one maternal chromosome13, and one paternal chromosome 13.

Submitted SNP (ss) 813773 was filled in with ddATP (lane 5) or ddCTP(lane 6) (FIG. 12). The calculated ratio for the nucleotides at thisheterozygous SNP, using the raw data, was 46:54. This ratio is within10% of the expected ratio of 50:50. Importantly, the ratio does notapproach the 66:33 ratio expected when there is an additional copy of achromosome.

After normalization to the internal control, the calculated ratio was41:59. Contrary to the expected result, normalization to the internalcontrol increased the discrepancy between the calculated ratio and thetheoretical ratio. This result may represent experimental error thatoccurred in aliquoting the DNA samples.

Also, it is possible that the restriction enzyme used to generate theoverhang, which was used as a template for the “fill-in” reaction,preferentially cut one DNA template over the other DNA template. The twotemplates differ, with respect to the nucleotide at the SNP site, andthis may influence the cutting. The primers can be designed such thatthe nucleotides adjacent to the cut site are the same, independent ofthe nucleotide at the SNP site (discussed further in the sectionentitled “Primer Design”).

SNP TSC 0198557, which is on chromosome 13, was filled in with ddTTP(lane 7) in one reaction and ddCTP (lane 8) in another (FIG. 12). Thecalculated ratio for the nucleotides at this SNP, using the raw data,was 55:45. After normalization to the internal control, the calculatedallele ratio of T:C was 49:51. The normalized ratio was closer to thetheoretical ratio of 50:50 for an individual with two copies ofchromosome 13.

SNP TSC 0197279, which is on chromosome 13, was filled in with ddTTP(lane 9) in one reaction and ddCTP (lane 10) in another (FIG. 12). Thecalculated ratio for the nucleotides at this SNP, using the raw data was53:47. After normalization to the internal control, the calculatedallele ratio of T:C was 50.7:49.3. This is consistent with thetheoretical ratio of 50:50 for an individual with only two copies ofchromosome 13.

The ratio for the nucleotides at two of the analyzed SNPs on chromosome13 was approximately 50:50. One SNP, ss813773, showed a ratio of 46:54,and when normalized to the internal control, the ratio was 41:59. Theseratios deviate from the expected 50:50, but at the same time, the ratiosare not indicative of an extra chromosome, which is indicated with aratio of 66:33. While the data from this particular SNP is inconclusive,it does not represent a false positive. No conclusion could be drawn onthe data from this SNP. However, the other two SNPs provided data thatindicated a normal number of chromosomes. It is preferable to analyzemultiple SNPs on a chromosome including but not limited to 1-5, 5-10,10-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-2000, 2000-3000, and greater than 3000.Preferably, the average of the ratios for a particular chromosome willbe used to determine the presence or absence of a chromosomalabnormality. However, it is still possible to analyze one locus ofinterest. In the event that inconclusive data is obtained, another locusof interest can be analyzed.

The individual from whom the DNA template was obtained had previouslybeen genotyped with trisomy 21, and the allele frequencies at SNPs onchromosome 21 indicate the presence of an additional chromosome 21. Theadditional chromosome contributes an additional nucleotide for each SNP,and thus alters the traditional 50:50 ratio at a heterozygous SNP. Theseresults are consistent for multiple SNPs, and are specific for thosefound on chromosome 21. The allele frequencies for SNPs on chromosome 13gave the expected ratios of approximately 50:50. These resultsdemonstrate that this method of SNP detection can be used to detectchromosomal abnormalities including but not limited to translocations,transversions, monosomies, trisomy 21, trisomy 18, trisomy 13, otheraneuploidies, deletions, additions, amplifications, translocations andrearrangements.

Example 6

Genomic DNA was obtained from four individuals after informed consentwas obtained. Six SNPs on chromosome 13 (TSC0837969, TSC0034767, TSC1130902, TSC0597888, TSC0195492, TSC0607185) were analyzed using thetemplate DNA. Information regarding these SNPs can be found at thefollowing website www.snp.chsl.org/snpsearch.shtml; website active as ofFeb. 11, 2003).

A single nucleotide labeled with one fluorescent dye was used togenotype the individuals at the six selected SNP sites. The primers weredesigned to allow the six SNPs to be analyzed in a single reaction.

Preparation of Template DNA

The template DNA was prepared from a 9 ml sample of blood obtained byvenipuncture from a human volunteer with informed consent. Template DNAwas isolated using the QIAmp DNA Blood Midi Kit supplied by QIAGEN(Catalog number 51183). The template DNA was isolated as perinstructions included in the kit.

Design of Primers

SNP TSC0837969 was amplified using the following primer set:

First primer: (SEQ ID NO: 30) 5′ GGGCTAGTCTCCGAATTCCACCTATCCTACCAAATGTC3′ Second primer: (SEQ ID NO: 31) 5′ TAGCTGTAGTTAGGGACTGTTCTGAGCAC 3′

The first primer had a biotin tag at the 5′ end and contained arestriction enzyme recognition site for EcoRI. The first primer wasdesigned to anneal 44 bases from the locus of interest. The secondprimer contained a restriction enzyme recognition site for BsmF I.

SNP TSC0034767 was amplified using the following primer set:

First primer: (SEQ ID NO: 32) 5′ CGAATGCAAGGCGAATTCGTTAGTAATAACACAGTGCA3′ Second primer: (SEQ ID NO: 33) 5′ AAGACTGGATCCGGGACCATGTAGAATAC 3′

The first primer had a biotin tag at the 5′ end and contained arestriction enzyme recognition site for EcoRI. The first primer wasdesigned to anneal 50 bases from the locus of interest. The secondprimer contained a restriction enzyme recognition site for BsmF I.

SNP TSC1130902 was amplified using the following primer set:

First primer: (SEQ ID NO: 34) 5′TCTAACCATTGCGAATTCAGGGCAAGGGGGGTGAGATC 3′ Second primer: (SEQ ID NO: 35)5′ TGACTTGGATCCGGGACAACGACTCATCC 3′

The first primer had a biotin tag at the 5′ end and contained arestriction enzyme recognition site for EcoRI. The first primer wasdesigned to anneal 60 bases from the locus of interest. The secondprimer contained a restriction enzyme recognition site for BsmF I.

SNP TSC0597888 was amplified using the following primer set:

First primer: (SEQ ID NO: 36) 5′ACCCAGGCGCCAGAATTCTTTAGATAAAGCTGAAGGGA 3′ Second primer: (SEQ ID NO: 37)5′ GTTACGGGATCCGGGACTCCATATTGATC 3′

The first primer had a biotin tag at the 5′ end and contained arestriction enzyme recognition site for EcoRI. The first primer wasdesigned to anneal 70 bases from the locus of interest. The secondprimer contained a restriction enzyme recognition site for BsmF I.

SNP TSC0195492 was amplified using the following primer set:

First primer: (SEQ ID NO: 38) 5′CGTTGGCTTGAGGAATTCGACCAAAAGAGCCAAGAGAASecond primer: (SEQ ID NO: 39) 5′ AAAAAGGGATCCGGGACCTTGACTAGGAC 3′

The first primer had a biotin tag at the 5′ end and contained arestriction enzyme recognition site for EcoRI. The first primer wasdesigned to anneal 80 bases from the locus of interest. The secondprimer contained a restriction enzyme recognition site for BsmF I.

SNP TSC0607185 was amplified using the following primer set:

First primer: (SEQ ID NO: 40) 5′ACTTGATTCCGTGAATTCGTTATCAATAAATCTTACAT 3′ Second primer: (SEQ ID NO: 41)5′ CAAGTTGGATCCGGGACCCAGGGCTAACC 3′

The first primer had a biotin tag at the 5′ end and contained arestriction enzyme recognition site for EcoRI. The first primer wasdesigned to anneal 90 bases from the locus of interest. The secondprimer contained a restriction enzyme recognition site for BsmF I.

All loci of interest were amplified from the template genomic DNA usingthe polymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and4,683,202, incorporated herein by reference). In this example, the lociof interest were amplified in separate reaction tubes but they couldalso be amplified together in a single PCR reaction. For increasedspecificity, a “hot-start” PCR was used. PCR reactions were performedusing the HotStarTaq Master Mix Kit supplied by QIAGEN (catalog number203443). The amount of template DNA and primer per reaction can beoptimized for each locus of interest but in this example, 40 ng oftemplate human genomic DNA and 5 μM of each primer were used. Fortycycles of PCR were performed. The following PCR conditions were used:

-   -   (1) 95° C. for 15 minutes and 15 seconds;    -   (2) 37° C. for 30 seconds;    -   (3) 95° C. for 30 seconds;    -   (4) 57° C. for 30 seconds;    -   (5) 95° C. for 30 seconds;    -   (6) 64° C. for 30 seconds;    -   (7) 95° C. for 30 seconds;    -   (8) Repeat steps 6 and 7 thirty nine (39) times;    -   (9) 72° C. for 5 minutes.

In the first cycle of PCR, the annealing temperature was about themelting temperature of the 3′ annealing region of the second primers,which was 37° C. The annealing temperature in the second cycle of PCRwas about the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the first primer, which was 57° C. The annealingtemperature in the third cycle of PCR was about the melting temperatureof the entire sequence of the second primer, which was 64° C. Theannealing temperature for the remaining cycles was 64° C. Escalating theannealing temperature from TM1 to TM2 to TM3 in the first three cyclesof PCR greatly improves specificity. These annealing temperatures arerepresentative, and the skilled artisan will understand the annealingtemperatures for each cycle are dependent on the specific primers used.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results. In this example, the first primer was designedto anneal at various distances from the locus of interest. The skilledartisan understands that the annealing location of the first primer canbe 5-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55,56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-105,106-110, 111-115, 116-120, 121-125, 126-130, 131-140, 1410-160,1610-180, 1810-200, 2010-220, 2210-240, 2410-260, 2610-280, 2810-300,3010-350, 3510-400, 4010-450, 450-500, or greater than 500 bases fromthe locus of interest.

Purification of Fragment of Interest

The PCR products were separated from the genomic template DNA. After thePCR reaction, ¼ of the volume of each PCR reaction from one individualwas mixed together in a well of a Streptawell, transparent, High-Bindplate from Roche Diagnostics GmbH (catalog number 1 645 692, as listedin Roche Molecular Biochemicals, 2001 Biochemicals Catalog). The firstprimers contained a 5′ biotin tag so the PCR products bound to theStreptavidin coated wells while the genomic template DNA did not. Thestreptavidin binding reaction was performed using a Thermomixer(Eppendorf) at 1000 rpm for 20 min. at 37° C. Each well was aspirated toremove unbound material, and washed three times with 1×PBS, with gentlemixing (Kandpal et al., Nucl. Acids Res. 18:1789-1795 (1990); Kaneoka etal., Biotechniques 10:30-34 (1991); Green et al, Nucl. Acids Res.18:6163-6164 (1990)).

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme BsmFI, which binds to the recognition site incorporated into the PCRproducts from the second primer. The digests were performed in theStreptawells following the instructions supplied with the restrictionenzyme. After digestion, the wells were washed three times with PBS toremove the cleaved fragments.

Incorporation of Labeled Nucleotide

The restriction enzyme digest with BsmF I yielded a DNA fragment with a5′ overhang, which contained the SNP site or locus of interest and a 3′recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide or nucleotides in the presence of a DNApolymerase.

Below, a schematic of the 5′ overhang for SNP TSC0837969 is shown. Theentire DNA sequence is not reproduced, only the portion to demonstratethe overhang (where R indicates the variable site).

5′ TTAA 3′ AATT R A C A Overhang position 1 2 3 4

The observed nucleotides for TSC0837969 on the 5′ sense strand (heredepicted as the top strand) are adenine and guanine. The third positionin the overhang on the antisense strand corresponds to cytosine, whichis complementary to guanine. As this variable site can be adenine orguanine, fluorescently labeled ddGTP in the presence of unlabeled dCTP,dTTP, and dATP was used to determine the sequence of both alleles. Thefill-in reactions for an individual homozygous for guanine, homozygousfor adenine or heterozygous are diagrammed below.

Homozygous for Guanine at TSC 0837969:

Allele 1 5′ TTAA G* 3′ AATT C A C A Overhang position 1 2 3 4 Allele 25′ TTAA G* 3′ AATT C A C A Overhang position 1 2 3 4

Labeled ddGTP is incorporated into the first position of the overhang.Only one signal is seen, which corresponds to the molecules filled inwith labeled ddGTP at the first position of the overhang.

Homozygous for Adenine at TSC 0837969:

Allele 1 5′ TTAA A T G* 3′ AATT T A C A Overhang position 1 2 3 4Allele 2 5′ TTAA A T G* 3′ AATT T A C A Overhang position 1 2 3 4

Unlabeled dATP is incorporated at position one of the overhang, andunlabeled dTTP is incorporated at position two of the overhang. LabeledddGTP was incorporated at position three of the overhang. Only onesignal will be seen; the molecules filled in with ddGTP at position 3will have a different molecular weight from molecules filled in atposition one, which allows easy identification of individuals homozygousfor adenine or guanine.

Heterozygous at TSC0837969:

Allele 1 5′ TTAA G* 3′ AATT C A C A Overhang position 1 2 3 4 Allele 25′ TTAA A T G* 3′ AATT T A C A Overhang position 1 2 3 4

Two signals will be seen; one signal corresponds to the DNA moleculesfilled in with ddGTP at position 1, and a second signal corresponding tomolecules filled in at position 3 of the overhang. The two signals canbe separated using any technique that separates based on molecularweight including but not limited to gel electrophoresis.

Below, a schematic of the 5′ overhang for SNP TSC0034767 is shown. Theentire DNA sequence is not reproduced, only the portion to demonstratethe overhang (where R indicates the variable site).

A C A R GTGT 3′ CACA 5′ 4 3 2 1 Overhang Position

The observed nucleotides for TSC0034767 on the 5′ sense strand (heredepicted as the top strand) are cytosine and guanine. The secondposition in the overhang corresponds to adenine, which is complementaryto thymidine. The third position in the overhang corresponds tocytosine, which is complementary to guanine. Fluorescently labeled ddGTPin the presence of unlabeled dCTP, dTTP, and dATP is used to determinethe sequence of both alleles.

In this case, the second primer anneals upstream of the locus ofinterest, and thus the fill-in reaction occurs on the anti-sense strand(here depicted as the bottom strand). Either the sense strand or theantisense strand can be filled in depending on whether the secondprimer, which contains the type IIS restriction enzyme recognition site,anneals upstream or downstream of the locus of interest.

Below, a schematic of the 5′ overhang for SNP TSC1130902 is shown. Theentire DNA sequence is not reproduced, only a portion to demonstrate theoverhang (where R indicates the variable site).

5′ TTCAT 3′ AAGTA R T C C Overhang position 1 2 3 4

The observed nucleotides for TSC1130902 on the 5′ sense strand (heredepicted as the top strand) are adenine and guanine. The second positionin the overhang corresponds to a thymidine, and the third position inthe overhang corresponds to cytosine, which is complementary to guanine.

Fluorescently labeled ddGTP in the presence of unlabeled dCTP, dTTP, anddATP is used to determine the sequence of both alleles.

Below, a schematic of the 5′ overhang for SNP TSC0597888 is shown. Theentire DNA sequence is not reproduced, only the portion to demonstratethe overhang (where R indicates the variable site).

T C T R ATTC 3′ TAAG 5′ 4 3 2 1 Overhang position

The observed nucleotides for TSC0597888 on the 5′ sense strand (heredepicted as the top strand) are cytosine and guanine. The third positionin the overhang corresponds to cytosine, which is complementary toguanine. Fluorescently labeled ddGTP in the presence of unlabeled dCTP,dTTP, and dATP is used to determine the sequence of both alleles.

Below, a schematic of the 5′ overhang for SNP TSC0607185 is shown. Theentire DNA sequence is not reproduced, only the portion to demonstratethe overhang (where R indicates the variable site).

C C T R TGTC 3′ ACAG 5′ 4 3 2 1 Overhang position

The observed nucleotides for TSC0607185 on the 5′ sense strand (heredepicted as the top strand) are cytosine and thymidine. In this case,the second primer anneals from the locus of interest, which allows theanti-sense strand to be filled in. The anti-sense strand (here depictedas the bottom strand) will be filled in with guanine or adenine.

The second position in the 5′ overhang is thymidine, which iscomplementary to adenine, and the third position in the overhangcorresponds to cytosine, which is complementary to guanine.Fluorescently labeled ddGTP in the presence of unlabeled dCTP, dTTP, anddATP is used to determine the sequence of both alleles.

Below, a schematic of the 5′ overhang for SNP TSC0195492 is shown. Theentire DNA sequence is not reproduced, only the portion to demonstratethe overhang.

5′ ATCT 3′ TAGA R A C A Overhang position 1 2 3 4

The observed nucleotides at this site are cytosine and guanine (heredepicted as the top strand). The second position in the 5′ overhang isadenine, which is complementary to thymidine, and the third position inthe overhang corresponds to cytosine, which is complementary to guanine.Fluorescently labeled ddGTP in the presence of unlabeled dCTP, dTTP, anddATP is used to determine the sequence of both alleles.

As demonstrated above, the sequence of both alleles of the six SNPs canbe determined by labeling with ddGTP in the presence of unlabeled dATP,dTTP, and dCTP. The following components were added to each fill inreaction: 1 μl of fluorescently labeled ddGTP, 0.5 μl of unlabeled dNTPs(40 μM), which contained all nucleotides except guanine, 2 μl of 10×sequenase buffer, 0.25 μl of Sequenase, and water as needed for a 20 μlreaction. The fill in reaction was performed at 40° C. for 10 min.Non-fluorescently labeled dNTP was purchased from Fermentas Inc.(Hanover, Md.). All other labeling reagents were obtained from Amersham(Thermo Sequenase Dye Terminator Cycle Sequencing Core Kit, US 79565).

After labeling, each Streptawell was rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments were then released from theStreptawells by digestion with the restriction enzyme EcoRI, accordingto the manufacturer's instructions that were supplied with the enzyme.Digestion was performed for 1 hour at 37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

After release from the streptavidin matrix, the sample was loaded into alane of a 36 cm 5% acrylamide (urea) gel (BioWhittaker MolecularApplications, Long Ranger Run Gel Packs, catalog number 50691). Thesample was electrophoresed into the gel at 3000 volts for 3 min. The gelwas run for 3 hours on a sequencing apparatus (Hoefer SQ3 Sequencer).The gel was removed from the apparatus and scanned on the Typhoon 9400Variable Mode Imager. The incorporated labeled nucleotide was detectedby fluorescence.

As shown in FIG. 11, the template DNA in lanes 1 and 2 for SNPTSC0837969 is homozygous for adenine. The following fill-in reaction wasexpected to occur if the individual was homozygous for adenine:

Homozygous for Adenine at TSC 0837969:

5′ TTAA A T G* 3′ AATT T A C A Overhang position 1 2 3 4

Unlabeled dATP was incorporated in the first position complementary tothe overhang. Unlabeled dTTP was incorporated in the second positioncomplementary to the overhang. Labeled ddGTP was incorporated in thethird position complementary to the overhang. Only one band was seen,which migrated at about position 46 of the acrylamide gel. Thisindicated that adenine was the nucleotide filled in at position one. Ifthe nucleotide guanine had been filled in, a band would be expected atposition 44.

However, the template DNA in lanes 3 and 4 for SNP TSC0837969 washeterozygous. The following fill-in reactions were expected if theindividual was heterozygous:

Heterozygous at TSC0837969:

Allele 1 5′ TTAA G* 3′ AATT C A C A Overhang position 1 2 3 4 Allele 25′ TTAA A T G* 3′ AATT T A C A Overhang position 1 2 3 4

Two distinct bands were seen; one band corresponds to the moleculesfilled in with ddGTP at position 1 complementary to the overhang (the Gallele), and the second band corresponds to molecules filled in withddGTP at position 3 complementary to the overhang (the A allele). Thetwo bands were separated based on the differences in molecular weightusing gel electrophoresis. One fluorescently labeled nucleotide ddGTPwas used to determine that an individual was heterozygous at a SNP site.This is the first use of a single nucleotide to effectively detect thepresence of two different alleles.

For SNP TSC0034767, the template DNA in lanes 1 and 3 is heterozygousfor cytosine and guanine, as evidenced by the two distinct bands. Thelower band corresponded to ddGTP filled in at position 1 complementaryto the overhang. The second band of slightly higher molecular weightcorresponded to ddGTP filled in at position 3, indicating that the firstposition in the overhang was filled in with unlabeled dCTP, whichallowed the polymerase to continue to incorporate nucleotides until itincorporated ddGTP at position 3 complementary to the overhang. Thetemplate DNA in lanes 2 and 4 was homozygous for guanine, as evidencedby a single band of higher molecular weight than if ddGTP had beenfilled in at the first position complementary to the overhang.

For SNP TSC1130902, the template DNA in lanes 1, 2, and 4 is homozygousfor adenine at the variable site, as evidenced by a single highermolecular weight band migrating at about position 62 on the gel. Thetemplate DNA in lane 3 is heterozygous at the variable site, asindicated by the presence of two distinct bands. The lower bandcorresponds to molecules filled in with ddGTP at position 1complementary to the overhang (the guanine allele). The higher molecularweight band corresponds to molecules filled in with ddGTP at position 3complementary to the overhang (the adenine allele).

For SNP TSC0597888, the template DNA in lanes 1 and 4 was homozygous forcytosine at the variable site; the template DNA in lane 2 washeterozygous at the variable site, and the template DNA in lane 3 washomozygous for guanine. The expected fill-in reactions are diagrammedbelow:

Homozygous for Cytosine:

Allele 1 T C T G ATTC 3′ G* A C TAAG 5′ 4 3 2 1 Overhang positionAllele 2 T C T G ATTC 3′ G* A C TAAG 5′ 4 3 2 1 Overhang position

Homozygous for Guanine:

Allele 1 T C T C ATTC 3′ G* TAAG 5′ 4 3 2 1 Overhang position Allele 2 TC T C ATTC 3′ G* TAAG 5′ 4 3 2 1 Overhang position

Heterozygous for Guanine/Cytosine:

Allele 1 T C T G ATTC 3′ G* A C TAAG 5′ 4 3 2 1 Overhang positionAllele 2 T C T C ATTC 3′ G* TAAG 5′ 4 3 2 1 Overhang position

Template DNA homozygous for guanine at the variable site displayed asingle band, which corresponded to the DNA molecules filled in withddGTP at position 1 complementary to the overhang. These DNA moleculeswere of lower molecular weight compared to the DNA molecules filled inwith ddGTP at position 3 of the overhang (see lane 3 for SNPTSC0597888). The DNA molecules differed by two bases in molecularweight.

Template DNA homozygous for cytosine at the variable site displayed asingle band, which corresponds to the DNA molecules filled in with ddGTPat position 3 complementary to the overhang. These DNA moleculesmigrated at a higher molecular weight than DNA molecules filled in withddGTP at position 1 (see lanes 1 and 4 for SNP TSC0597888).

Template DNA heterozygous at the variable site displayed two bands; oneband corresponded to the DNA molecules filled in with ddGTP at position1 complementary to the overhang and was of lower molecular weight, andthe second band corresponded to DNA molecules filled in with ddGTP atposition 3 complementary to the overhang, and was of higher molecularweight (see lane 3 for SNP TSC0597888).

For SNP TSC0195492, the template DNA in lanes 1 and 3 was heterozygousat the variable site, which was demonstrated by the presence of twodistinct bands. The template DNA in lane 2 was homozygous for guanine atthe variable site. The template DNA in lane 4 was homozygous forcytosine. Only one band was seen in lane 4 for this SNP, and it had ahigher molecular weight than the DNA molecules filled in with ddGTP atposition 1 complementary to the overhang (compare lanes 2, 3 and 4).

The observed alleles for SNP TSC0607185 are reported as cytosine orthymidine. For consistency, the SNP consortium denotes the observedalleles as they appear in the sense strandwww.snp.cshl.org/shpsearch.shtml; website active as of Feb. 11, 2003).For this SNP, the second primer annealed upstream of the locus ofinterest, which allowed the fill-in reaction to occur on the antisensestrand after digestion with BsmF I.

The template DNA in lanes 1 and 3 was heterozygous; the template DNA inlane 2 was homozygous for thymidine, and the template DNA in lane 4 washomozygous for cytosine. The antisense strand was filled in with ddGTP,so the nucleotide on the sense strand corresponded to cytosine.

Molecular weight markers can be used to identify the positions of theexpected bands. Alternatively, for each SNP analyzed, a knownheterozygous sample can be used, which will identify precisely theposition of the two expected bands.

As demonstrated in FIG. 11, one nucleotide labeled with one fluorescentdye can be used to determine the identity of a variable site includingbut not limited to SNPs and single nucleotide mutations. Typically, todetermine if an individual is homozygous or heterozygous at a SNP site,multiple reactions are performed using one nucleotide labeled with onedye and a second nucleotide labeled with a second dye. However, thisintroduces problems in comparing results because the two dyes havedifferent quantum coefficients. Even if different nucleotides arelabeled with the same dye, the quantum coefficients are different. Theuse of a single nucleotide labeled with one dye eliminates any errorsfrom the quantum coefficients of different dyes.

In this example, fluorescently labeled ddGTP was used. However, themethod is applicable for a nucleotide tagged with any signal generatingmoiety including but not limited to radioactive molecule, fluorescentmolecule, antibody, antibody fragment, hapten, carbohydrate, biotin,derivative of biotin, phosphorescent moiety, luminescent moiety,electrochemiluminescent moiety, chromatic moiety, and moiety having adetectable electron spin resonance, electrical capacitance, dielectricconstant or electrical conductivity. In addition, labeled ddATP, ddTTP,or ddCTP can be used.

The above example used the third position complementary to the overhangas an indicator of the second allele. However, the second or fourthposition of the overhang can be used as well (see Section onIncorporation of Nucleotides). Furthermore, the overhang was generatedwith the type IIS enzyme BsmF I; however any enzymes that cuts DNA at adistance from its binding site can be used including but not limited tothe enzymes listed in Table I.

Also, in the above example, the nucleotide immediately preceding the SNPsite was not a guanine on the strand that was filled in. This eliminatedany effects of the alternative cutting properties of the type IISrestriction enzyme to be removed. For example, at SNP TSC0837969, thenucleotide from the SNP site on the sense strand was an adenine. If BsmFI displayed alternate cutting properties, the following overhangs wouldbe generated for the adenine allele and the guanine allele:

G allele - 11/15 Cut 5′ TTA 3′ AAT T C A C Overhang position 0 1 2 3G allele after fill-in 5′ TTA A G* 3′ AAT T C A C Overhang position 0 12 3 A allele 11/15 Cut 5′ TTA 3′ AAT T T A C Overhang position 0 1 2 3A allele after fill-in 5′ TTA A A T G* 3′ AAT T T A C Overhang position0 1 2 3

For the guanine allele, the first position in the overhang would befilled in with dATP, which would allow the polymerase to incorporateddGTP at position 2 complementary to the overhang. There would be nodetectable difference between molecules cut at the 10/14 position ormolecules cut at the 11/15 position.

For the adenine allele, the first position complementary to the overhangwould be filled in with dATP, the second position would be filled inwith dATP, the third position would be filled in with dTTP, and thefourth position would be filled in with ddGTP. There would be nodifference in the molecular weights between molecules cut at 10/14 ormolecules cut at 11/15. The only differences would correspond to whetherthe DNA molecules contained an adenine at the variable site or a guanineat the variable site.

As seen in FIG. 11, positioning the annealing region of the first primerallows multiple SNPs to be analyzed in a single lane of a gel. Also,when using the same nucleotide with the same dye, a single fill-inreaction can be performed. In this example, 6 SNPs were analyzed in onelane. However, any number of SNPs including but not limited to 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 30-40, 40-50, 51-60, 61-70, 71-80,81-100, 101-120, 121-140, 141-160, 161-180, 181-200, and greater than200 can be analyzed in a single reaction.

Furthermore, one labeled nucleotide used to detect both alleles can bemixed with a second labeled nucleotide used to detect a different set ofSNPs provided that neither of the nucleotides that are labeled occurimmediately before the variable site (complementary to nucleotide atposition 0 of the 11/15 cut). For example, suppose SNP X can be guanineor thymidine at the variable site and has the following 5′ overhanggenerated after digestion with BsmF I:

SNP X 10/14 5′ TTGAC G allele 3′AACTG C A C T Overhang position 1 2 3 4SNP X 11/15 5′ TTGA G allele 3′AACT G C A C Overhang position 0 1 2 3SNP X 10/14 5′ TTGAC T allele 3′AACTG A A C T Overhang position 1 2 3 4SNP X 11/15 5′ TTGA T allele 3′AACT G A A C Overhang position 0 1 2 3

After the fill-in reaction with labeled ddGTP, unlabeled dATP, dCTP, anddTTP, the following molecules would be generated:

SNP X 10/14 5′ TTGAC G* G allele 3′AACTG C A C T Overhang position 1 2 34 SNP X 11/15 5′ TTGA C G* G allele 3′AACT G C A C Overhang position 0 12 3 SNP X 10/14 5′ TTGAC T T G* T allele 3′AACTG A A C TOverhang position 1 2 3 4 SNP X 11/15 5′ TTGA C T T G* T allele 3′AACT GA A C Overhang position 0 1 2 3

Now suppose SNP Y can be adenine or thymidine at the variable site, andhas the following 5′ overhangs generated after digestion with BsmF I.

SNP Y 10/14 5′ GTTT A allele 3′ CAAA T G T A Overhang position 1 2 3 4SNP Y 11/15 5′ GTT A allele 3′ CAA A T G T Overhang position 0 1 2 3SNP Y 10/14 5′ GTTT T allele 3′ CAAA A G T A Overhang position 1 2 3 4SNP Y 11/15 5′ GTT T allele 3′ CAA A A G T Overhang position 0 1 2 3

After fill-in with labeled ddATP and unlabeled dCTP, dGTP, and dTTP, thefollowing molecules would be generated:

SNP Y 10/14 5′ GTTT A* A allele 3′ CAAA T G T A Overhang position 1 2 34 SNP Y 11/15 5′ GTTT T A* A allele 3′ CAA A T G T Overhang position 0 12 3 SNP Y 10/14 5′ GTTT T C A* T allele 3′ CAAA A G T AOverhang position 1 2 3 4 SNP Y 11/15 5′ GTT T T C A* T allele 3′ CAA AA G T Overhang position 0 1 2 3

In this example, labeled ddGTP and labeled ddATP are used to determinethe identity of both alleles of SNP X and SNP Y respectively. Thenucleotide immediately preceding (the complementary nucleotide toposition 0 of the overhang from the 11/15 cut SNP X is not guanine oradenine on the strand that is filled-in. Likewise, the nucleotideimmediately preceding SNPY is not guanine or adenine on the strand thatis filled-in. This allows the fill-in reaction for both SNPs to occur ina single reaction with labeled ddGTP, labeled ddATP, and unlabeled dCTPand dTTP. This reduces the number of reactions that need to be performedand increases the number of SNPs that can be analyzed in one reaction.

The first primers for each SNP can be designed to anneal at differentdistances from the locus of interest, which allows the SNPs to migrateat different positions on the gel. For example, the first primer used toamplify SNP X can anneal at 30 bases from the locus of interest, and thefirst primer used to amplify SNP Y can anneal at 35 bases from the locusof interest. Also, the nucleotides can be labeled with fluorescent dyesthat emit at spectrums that do not overlap. After running the gel, thegel can be scanned at one wavelength specific for one dye. Only thosemolecules labeled with that dye will emit a signal. The gel then can bescanned at the wavelength for the second dye. Only those moleculeslabeled with that dye will emit a signal. This method allows maximumcompression for the number of SNPs that can be analyzed in a singlereaction.

In this example, the nucleotide preceding the variable site on thestrand that was filled-in was not adenine or guanine, and the nucleotidefollowing the variable site can not be adenine or guanine on the sensestrand. This method can work with any combination of labelednucleotides, and the skilled artisan would understand which labelingreactions can be mixed and those that can not. For instance, if one SNPis labeled with thymidine and a second SNP is labeled with cytosine, theSNPs can be labeled in a single reaction if the nucleotide immediatelypreceding each variable site is not thymidine or cytosine on the sensestrand and the nucleotide immediately after the variable site is notthymidine or cytosine on the sense strand.

This method allows the signals from one allele to be compared to thesignal from a second allele without the added complexity of determiningthe degree of alternate cutting, or having to correct for the quantumcoefficients of the dyes. This method is especially useful when tryingto quantitate a ratio for one allele to another. For example, thismethod is useful for detecting chromosomal abnormalities. The ratio ofalleles at a heterozygous site is expected to be about 1:1 (one A alleleand one G allele). However, if an extra chromosome is present the ratiois expected to be about 1:2 (one A allele and 2 G alleles or 2 A allelesand 1 G allele). This method is especially useful when trying to detectfetal DNA in the presence of maternal DNA.

In addition, this method is useful for detecting two genetic signals inone sample. For example, this method can detect mutant cells in thepresence of wild type cells (see Example 5). If a mutant cell contains amutation in the DNA sequence of a particular gene, this method can beused to detect both the mutant signal and the wild type signal. Thismethod can be used to detect the mutant DNA sequence in the presence ofthe wild type DNA sequence. The ratio of mutant DNA to wild type DNA canbe quantitated because a single nucleotide labeled with one signalgenerating moiety is used.

Example 7

Non-invasive methods for the detection of various types of cancer havethe potential to reduce morbidity and mortality from the disease.Several techniques for the early detection of colorectal tumors havebeen developed including colonoscopy, barium enemas, and sigmoidoscopy;however the techniques are limited in use because they are invasive,which causes a low rate of patient compliance. Non-invasive genetictests may be useful in identifying early stage colorectal tumors.

In 1991, researchers identified the Adenomatous Polyposis Coli gene(APC), which plays a critical role in the formation of colorectal tumors(Kinzler et al., Science 253:661-665, 1991). The APC gene resides onchromosome 5q21-22 and a total of 15 exons code for an RNA molecule of8529 nucleotides, which produces a 300 Kd APC protein. The protein isexpressed in numerous cell types and is essential for cell adhesion.

Mutations in the APC gene generally initiate colorectal neoplasia (Tsao,J. et al., Am, J. Pathol. 145:531-534, 1994). Approximately 95% of themutations in the APC gene result in nonsense/frameshift mutations. Themost common mutations occur at codons 1061 and 1309; mutations at thesecodons account for ⅓ of all germline mutations. With regard to somaticmutations, 60% occur within codons 1286-1513, which is about 10% of thecoding sequence. This region is termed the mutation Cluster Region(MCR), Numerous types of mutations have been identified in the APC geneincluding nucleotide substitutions (see Table VII), splicing errors (seeTable VIII), small deletions (see Table IX), small insertions (see TableX), small insertions/deletions (see Table XI), gross deletions (seeTable XII), gross insertions (see Table XIII), and complexrearrangements (see Table XIV).

Researchers have attempted to identify cells harboring mutations in theAPC gene in stool samples (Traverso, G. et al., New England Journal ofMedicine, Vol 346:311-320, 2002). While APC mutations are found innearly all tumors, about 1 in 250 cells in the stool sample has amutation in the APC gene; most of the cells are normal cells that havebeen shed into the feces. Furthermore, human DNA represents aboutone-billionth of the total DNA found in stool samples; the majority ofDNA is bacterial. The technique employed by Traverso et al. only detectsmutations that result in a truncated protein.

As discussed above, numerous mutations in the APC gene have beenimplicated in the formation of colorectal tumors. Thus, a need stillexists for a highly sensitive, non-invasive technique for the detectionof colorectal tumors. Below, methods are described for detection of twomutations in the APC gene. However, any number of mutations can beanalyzed using the methods described herein.

Preparation of Template DNA

The template DNA is purified from a sample containing colon cellsincluding but not limited to a stool sample. The template DNA ispurified using the procedures described by Ahlquist et al.(Gastroenterology, 119:1219-1227, 2000). If stool samples are frozen,the samples are thawed at room temperature, and homogenized with anExactor stool shaker (Exact Laboratories, Maynard, Mass.) Followinghomogenization, a 4 gram stool equivalent of each sample is centrifugedat 2536×g for 5 minutes. The samples are centrifuged a second time at16, 500×g for 10 minutes. Supernatants are incubated with 20 μl of RNase(0.5 mg per milliliter) for 1 hour at 37° C. DNA is precipitated with1/10 volume of 3 mol of sodium acetate per liter and an equal volume ofisopropanol. The DNA is dissolved in 5 ml of TRIS-EDTA (0.01 mol of Trisper liter (pH 7.4) and 0.001 mole of EDTA per liter.

Design of Primers

To determine if a mutation resides at codon 1370, the following primersare used:

First primer: (SEQ ED NO: 42) 5′GTGCAAAGGCCTGAATTCCCAGGCACAAAGCTGTTGAA 3′ Second primer: (SEQ ID NO: 43)5′ TGAAGCGAACTAGGGACTCAGGTGGACTT

The first primer contains a biotin tag at the extreme 5′ end, and thenucleotide sequence for the restriction enzyme EcoRI. The second primercontains the nucleotide sequence for the restriction enzyme BsmF I.

To determine if a small deletion exists at codon 1302, the followingprimers are used:

First primer: (SEQ ID NO: 44) 5′GATTCCGTAAACGAATTCAGTTCATTATCATCTTTGTC 3′ Second primer: (SEQ ID NO: 45)5′ CCATTGTTAAGCGGGACTTCTGCTATTTG 3′

The first primer has a biotin tag at the 5′ end and contains arestriction enzyme recognition site for EcoRI. The second primercontains a restriction enzyme recognition site for BsmF I,

PCR Reaction

The loci of interest are amplified from the template genomic DNA usingthe polymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and4,683,202, incorporated herein by reference). The loci of interest areamplified in separate reaction tubes; they can also be amplifiedtogether in a single PCR reaction. For increased specificity, a“hot-start” PCR reaction is used, e.g. by using the HotStarTaq MasterMix Kit supplied by QIAGEN (catalog number 203443). The amount oftemplate DNA and primer per reaction are optimized for each locus ofinterest but in this example, 40 ng of template human genomic DNA and 5μM of each primer are used. Forty cycles of PCR are performed. Thefollowing PCR conditions are used:

-   -   (1) 95° C. for 15 minutes and 15 seconds;    -   (2) 37° C. for 30 seconds;    -   (3) 95° C. for 30 seconds;    -   (4) 57° C. for 30 seconds;    -   (5) 95° C. for 30 seconds;    -   (6) 64° C. for 30 seconds;    -   (7) 95° C. for 30 seconds;    -   (8) Repeat steps 6 and 7 thirty nine (39) times;    -   (9) 72° C. for 5 minutes.

In the first cycle of PCR, the annealing temperature is about themelting temperature of the 3′ annealing region of the second primers,which is 37° C. The annealing temperature in the second cycle of PCR isabout the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the first primer, which is 57° C. The annealingtemperature in the third cycle of PCR is about the melting temperatureof the entire sequence of the second primer, which is 64° C. Theannealing temperature for the remaining cycles is 64° C. Escalating theannealing temperature from TM1 to TM2 to TM3 in the first three cyclesof PCR greatly improves specificity. These annealing temperatures arerepresentative, and the skilled artisan understands that the annealingtemperatures for each cycle are dependent on the specific primers used.

The temperatures and times for denaturing, annealing, and extension, areoptimized by trying various settings and using the parameters that yieldthe best results.

Purification of Fragment of Interest

The PCR products are separated from the genomic template DNA. Each PCRproduct is divided into four separate reaction wells of a Streptawell,transparent, High-Bind plate from Roche Diagnostics GmbH (catalog number1 645 692, as listed in Roche Molecular Biochemicals, 2001 BiochemicalsCatalog). The first primers contain a 5′ biotin tag so the PCR productsbound to the Streptavidin coated wells while the genomic template DNAdoes not. The streptavidin binding reaction is performed using aThermomixer (Eppendorf) at 1000 rpm for 20 min. at 37° C. Each well isaspirated to remove unbound material, and washed three times with 1×PBS,with gentle mixing (Kandpal et al., Nucl. Acids Res. 18:1789-1795(1990); Kaneoka et al., Biotechniques 10:30-34 (1991); Green et al.,Nucl. Acids Res. 18:6163-6164 (1990)).

Alternatively, the PCR products are placed into a single well of astreptavidin plate to perform the nucleotide incorporation reaction in asingle well.

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products are digested with the restriction enzyme BsmFI (New England Biolabs catalog number R0572S), which binds to therecognition site incorporated into the PCR products from the secondprimer. The digests are performed in the Streptawells following theinstructions supplied with the restriction enzyme. After digestion withthe appropriate restriction enzyme, the wells are washed three timeswith PBS to remove the cleaved fragments.

Incorporation of Labeled Nucleotide

The restriction enzyme digest described above yields a DNA fragment witha 5′ overhang, which contains the locus of interest and a 3′ recessedend. The 5′ overhang functions as a template allowing incorporation of anucleotide or nucleotides in the presence of a DNA polymerase.

For each locus of interest, four separate fill in reactions areperformed; each of the four reactions contains a different fluorescentlylabeled ddNTP (ddATP, ddTTP, ddGTP, or ddCTP). The following componentsare added to each fill in reaction: 1 μl of a fluorescently labeledddNTP, 0.5 μl of unlabeled ddNTPs (40 μM), which contains allnucleotides except the nucleotide that is fluorescently labeled, 2 μl of10× sequenase buffer, 025 μl of Sequenase, and water as needed for a 20μl reaction. The fill are performed in reactions at 40° C. for 10 min.Non-fluorescently labeled ddNTP are purchased from Fermentas Inc.(Hanover, Md.). All other labeling reagents are obtained from Amersham(Thermo Sequenase Dye Terminator Cycle Sequencing Core Kit, US 79565).In the presence of fluorescently labeled ddNTPs, the 3′ recessed end isextended by one base, which corresponds to the locus of interest.

A mixture of labeled ddNTPs and unlabeled dNTPs also can be used for thefill-in reaction. The “fill in” conditions are as described above exceptthat a mixture containing 40 μM unlabeled dNTPs, 1 μl fluorescentlylabeled ddATP, 1 μl fluorescently labeled ddTTP, 1 μl fluorescentlylabeled ddCTP, and 1 μl ddGTP are used. The fluorescent ddNTPs areobtained from Amersham (Thermo Sequenase Dye Terminator Cycle SequencingCore Kit, US 79565; Amersham does not publish the concentrations of thefluorescent nucleotides). The locus of interest is digested with therestriction enzyme BsmF I, which generates a 5′ overhang of four bases.If the first nucleotide incorporated is a labeled ddNTP, the 3′ recessedend is filled in by one base, allowing detection of the locus ofinterest. However, if the first nucleotide incorporated is a dNTP, thepolymerase continues to incorporate nucleotides until a ddNTP is filledin. For example, the first two nucleotides may be filled in with dNTPs,and the third nucleotide with a ddNTP, allowing detection of the thirdnucleotide in the overhang. Thus, the sequence of the entire 5′ overhangis determined, which increases the information obtained from each SNP orlocus of interest. This type of fill in reaction is especially usefulwhen detecting the presence of insertions, deletions, insertions anddeletions, rearrangements, and translocations.

Alternatively, one nucleotide labeled with a single dye is used todetermine the sequence of the locus of interest. See Example 6. Thismethod eliminates any potential errors when using different dyes, whichhave different quantum coefficients.

After labeling, each Streptawell is rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments are released from the Streptawellsby digesting with the restriction enzyme EcoRI, according to themanufacturer's instructions that are supplied with the enzyme. Thedigestion is performed for 1 hour at 37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

After release from the streptavidin matrix, the sample is loaded into alane of a 36 cm 5% acrylamide (urea) gel (BioWhittaker MolecularApplications, Long Ranger Run Gel Packs, catalog number 50691). Thesample is electrophoresed into the gel at 3000 volts for 3 min. The gelis run for 3 hours using a sequencing apparatus (Hoefer SQ3 Sequencer).The incorporated labeled nucleotide is detected by fluorescence.

To determine if any cells contain mutations at codon 1370 of the APCgene when separate fill-in reactions are performed, the lanes of the gelthat correspond to the fill-in reaction for ddATP and ddTTP areanalyzed. If only normal cells are present, the lane corresponding tothe fill in reaction with ddATP is a bright signal. No signal isdetected for the “fill-in” reaction with ddTTP. However, if the patientsample contains cells with mutations at codon 1370 of the APC gene, thelane corresponding to the fill in reaction with ddATP is a brightsignal, and a signal is detected from the lane corresponding to the fillin reaction with ddTTP. The intensity of the signal from the lanecorresponding to the fill in reaction with ddTTP is indicative of thenumber of mutant cells in the sample.

Alternatively, one labeled nucleotide is used to determine the sequenceof the alleles at codon 1370 of the APC gene. At codon 1370, the normalsequence is AAA, which codes for the amino acid lysine. However, anucleotide substitution has been identified at codon 1370, which isassociated with colorectal tumors. Specifically, a change from A to T(AAA-TAA) typically is found at codon 1370, which results in a stopcodon. A single fill-in reaction is performed using labeled ddATP, andunlabeled dTTP, dCTP, and dGTP. A single nucleotide labeled with onefluorescent dye is used to determine the presence of both the normal andmutant DNA sequence that codes for codon 1370. The relevant DNA sequenceis depicted below with the sequence corresponding to codon 1370 in bold:

5′ CCCAAAAGTCCACCTGA (SEQ ID NO: 46) 3′ GGGTTTTCAGGTGGACT(SEQ ID NO: 47)

After digest with BsmF I, the following overhang is produced:

5′ CCC 3′ GGG T T T T Overhang position 1 2 3 4

If the patient sample has no cells harboring a mutation at codon 1370,one signal is seen corresponding to incorporation of labeled ddATP.

5′ CCC A* 3′ GGG T T T T Overhang position 1 2 3 4

However, if the patient sample has cells with mutations at codon 1370 ofthe APC gene, one signal is seen, which corresponds to the normalsequence at codon 1370, and a second signal is seen, which correspondsto the mutant sequence at codon 1370. The signals clearly are identifiedas they differ in molecular weight.

Overhang of normal DNA sequence: CCC GGG T T T T Overhang position 1 2 34 Normal DNA sequence after fill-in: CCC A* GGG T T T TOverhang position 1 2 3 4 Overhang of mutant DNA sequence: CCC GGG A T TT Overhang position 1 2 3 4 Mutant DNA sequence after fill-in: CCC T A*GGG A T T T Overhang position 1 2 3 4

Two signals are seen when the mutant allele is present. The mutant DNAmolecules are filled in one base after the wild type DNA molecules. Thetwo signals are separated using any method that discriminates based onmolecular weight. One labeled nucleotide (ddATP) is used to detect thepresence of both the wild type DNA sequence and the mutant DNA sequence.This method of labeling reduces the number of reactions that need to beperformed and allows accurate quantitation for the number of mutantcells in the patient sample. The number of mutant cells in the sample isused to determine patient prognosis, the degree and the severity of thedisease. This method of labeling eliminates the complications associatedwith using different dyes, which have distinct quantum coefficients.This method of labeling also eliminates errors associated with pipettingreactions.

To determine if any cells contain mutations at codon 1302 of the APCgene when separate fill-in reactions are performed, the lanes of the gelthat correspond to the fill-in reaction for ddTTP and ddCTP areanalyzed. The normal DNA sequence is depicted below with sequence codingfor codon 1302 in bold type-face.

Normal Sequence: 5′ ACCCTGCAAATAGCAGAA (SEQ ID NO: 48) 3′TGGGACGTTTATCGTCTT (SEQ ID NO: 49)

After digest, the following 5′ overhang is produced:

5′ ACCC 3′ TGGG A C G T Overhang position 1 2 3 4

After the fill-in reaction, labeled ddTTP is incorporated.

5′ ACCC T* 3′ TGGG A C G T Overhang position 1 2 3 4

A deletion of a single base of the APC sequence, which typically codesfor codon 1302, has been associated with colorectal tumors. The mutantDNA sequence is depicted below with the relevant sentience in bold:

Mutant Sequence: 5′ ACCCGCAAATAGCAGAA (SEQ ID NO: 50) 3′TGGGCGTTTATCGTCTT (SEQ ID NO: 51)

After Digest:

5′ ACC 3′ TGG G C G T Overhang position 1 2 3 4

After Fill-in:

5′ ACC C* 3′ TGG G C G T Overhang position 1 2 3 4

If there are no mutations in the APC gene, signal is not detected forthe fill in reaction with ddCTP*, but a bright signal is detected forthe fill-in reaction with ddTTP*. However, if there are cells in thepatient sample that have mutations in the APC gene, signals are seen forthe fill-in reactions with ddCTP*and ddTTP*.

Alternatively, a single fill-in reaction is performed using a mixturecontaining unlabeled dNTPs, fluorescently labeled ddATP, fluorescentlylabeled ddTTP, fluorescently labeled ddCTP, and fluorescently labeledddGTP. If there is no deletion, labeled ddTTP is incorporated.

5′ ACCC T* 3′ TGGG A C G T Overhang position 1 2 3 4

However, if the T has been deleted, labeled ddCTP* is incorporated.

5′ ACCC* 3′ TGGG C G T Overhang position 1 2 3 4

The two signals are separated by molecular weight because of thedeletion of the thymidine nucleotide. If mutant cells are present, twosignals are generated in the same lane but are separated by a singlebase pair (this principle is demonstrated in FIG. 9D). The deletioncauses a change in the molecular weight of the DNA fragments, whichallows a single fill in reaction to be used to detect the presence ofboth normal and mutant cells.

In the above example, methods for the detection of a nucleotidesubstitution and a small deletion are described. However, the methodscan be used for the detection of any type of mutation including but notlimited to nucleotide substitutions (see Table VII), splicing errors(see Table VIII), small deletions (see Table IX), small insertions (seeTable X), small insertions/deletions (see Table XI), gross deletions(see Table XII), gross insertions (see Table XIII), and complexrearrangements (see Table XIV).

In addition, the above-described methods are used for the detection ofany type of disease including but not limited to those listed in TableIV. Furthermore, any type of mutant gene is detected using theinventions described herein including but not limited to the genesassociated with the diseases listed in Table IV, BRCA1, BRCA2, MSH6,MSH2, MLH1, RET, PTEN, ATM, H-RAS, p53, ELAC2, CDH1, APC, AR, PMS2,MLH3, CYP1A1, GSTP1, GSTM1, AXIN2, CYP19, MET, NAT1, CDKN2A, NQ01, trc8,RAD51, PMS1, TGFBR2, VHL, MC4R, POMC, NROB2, UCP2, PCSK1, PPARG, ADRB2,UCP3, glur1, cart, SORBS1, LEP, LEPR, SIM1, TNF, IL-6, IL-1, IL-2, IL-3,IL1A, TAP2, THPO, THRB, NBS1, RBM15, LIF, MPL, RUNX1, Her-2,glucocorticoid receptor, estrogen receptor, thyroid receptor, p21, p27,K-RAS, N-RAS, retinoblastoma protein, Wiskott-Aldrich (WAS) gene, FactorV Leiden, Factor II (prothrombin), methylene tetrahydrofolate reductase,cystic fibrosis, LDL receptor, HDL receptor, superoxide dismutase gene,SHOX gene, genes involved in nitric oxide regulation, genes involved incell cycle regulation, tumor suppressor genes, oncogenes, genesassociated with neurodegeneration, genes associated with obesity.Abbreviations correspond to the proteins as listed on the Human GeneMutation Database, which is incorporated herein by referencewww.archive.uwcm.ac.uk./uwcm; website address active as of Feb. 12,2003).

The above example demonstrates the detection of mutant cells and mutantalleles from a fecal sample. However, the methods described herein areused for detection of mutant cells from any biological sample includingbut not limited to blood sample, serum sample, plasma sample, urinesample, spinal fluid, lymphatic fluid, semen, vaginal secretion, asciticfluid, saliva, mucosa secretion, peritoneal fluid, fecal sample, bodyexudates, breast fluid, lung aspirates, cells, tissues, individual cellsor extracts of the such sources that contain the nucleic acid of thesame, and subcellular structures such as mitochondria or chloroplasts.In addition, the methods described herein are used for the detection ofmutant cells and mutated DNA from any number of nucleic acid containingsources including but not limited to forensic, food, archeological,agricultural or inorganic samples.

The above example is directed to detection of mutations in the APC gene.However, the inventions described herein are used for the detection ofmutations in any gene that is associated with or predisposes to disease(see Table XV).

For example, hypermethylation of the glutathione S-transferase P1(GSTP1) promoter is the most common DNA alteration in prostrate cancer.The methylation state of the promoter is determined using sodiumbisulfite and the methods described herein.

Treatment with sodium bisulfite converts unmethylated cytosine residuesinto uracil, and leaving the methylated cytosines unchanged. Using themethods described herein, a first and second primer are designed toamplify the regions of the GSTP1 promoter that are often methylated.Below, a region of the GSTP1 promoter is shown prior to sodium bisulfitetreatment:

Before Sodium Bisulfite Treatment:

5′ ACCGCTACA 3′ TGGCGATCA

Below, a region of the GSTP1 promoter is shown after sodium bisulfitetreatment, PCR amplification, and digestion with the type IISrestriction enzyme BsmF I;

Unmethylated 5′ ACC 3′ TGG U G A T Overhang position 1 2 3 4 Methylated5′ ACC 3′ TGG C G A T Overhang position 1 2 3 4

Labeled ddATP, unlabeled dCTP, dGTP, and dTTP are used to fill-in the 5′overhangs. The following molecules are generated:

Unmethylated 5′ ACC A* 3′ TGG U G A T Overhang position 1 2 3 4Methylated 5′ ACC G C T A* 3′ TGG C G A T Overhang position 1 2 3 4

Two signals are seen; one corresponds to DNA molecules filled in withddATP at position one complementary to the overhang (unmethylated), andthe other corresponds to the DNA molecules filled in with ddATP atposition 4 complementary to the overhang (methylated). The two signalsare separated based on molecular weight. Alternatively, the fill-inreactions are performed in separate reactions using labeled ddGTP in onereaction and labeled ddATP in another reaction.

The methods described herein are used to screen for prostate cancer andalso to monitor the progression and severity of the disease. The use ofa single nucleotide to detect both the methylated and unmethylatedsequences allows accurate quantitation and provides a high level ofsensitivity for the methylated sequences, which is a useful tool forearlier detection of the disease.

The information contained in Tables VII-XIV was obtained from the HumanGene Mutation Database. With the information provided herein, theskilled artisan will understand how to apply these methods fordetermining the sequence of the alleles for any gene. A large number ofgenes and there associated mutations can be found at the followingwebsite: www.archive.uwcm.ac.uk./uwcm.

TABLE VII NUCLEOTIDE SUBSTITUTIONS Co- Amino don Nucleotide acidPhenotype 99 CGG-TGG Arg-Trp Adenomatous polyposis coli 121 AGA-TGAArg-Term Adenomatous polyposis coli 157 TGG-TAG Trp-TermAdenomatous polyposis coli 159 TAC-TAG Tyr-TermAdenomatous polyposis coli 163 CAG-TAG Gln-TermAdenomatous polyposis coli 168 AGA-TGA Arg-TermAdenomatous polyposis coli 171 AGT-ATT Ser-IleAdenomatous polyposis coli 181 CAA-TAA Gln-TermAdenomatous polyposis coli 190 GAA-TAA Glu-TermAdenomatous polyposis coli 202 GAA-TAA Glu-TermAdenomatous polyposis coli 208 CAG-CGG Gln-ArgAdenomatous polyposis coli 208 CAG-TAG Gln-TermAdenomatous polyposis coli 213 CGA-TGA Arg-TermAdenomatous polyposis coli 215 CAG-TAG Gln-TermAdenomatous polyposis coli 216 CGA-TGA Arg-TermAdenomatous polyposis coli 232 CGA-TGA Arg-TermAdenomatous polyposis coli 233 CAG-TAG Gln-TermAdenomatous polyposis coli 247 CAG-TAG Gln-TermAdenomatous polyposis coli 267 GGA-TGA Gly-TermAdenomatous polyposis coli 278 CAG-TAG Gln-TermAdenomatous polyposis coli 280 TCA-TGA Ser-TermAdenomatous polyposis coli 280 TCA-TAA Ser-TermAdenomatous polyposis coli 283 CGA-TGA Arg-TermAdenomatous polyposis coli 302 CGA-TGA Arg-TermAdenomatous polyposis coli 332 CGA-TGA Arg-TermAdenomatous polyposis coli 358 CAG-TAG Gln-TermAdenomatous polyposis coli 405 CGA-TGA Arg-TermAdenomatous polyposis coli 414 CGC-TGC Arg-CysAdenomatous polyposis coli 422 GAG-TAG Glu-TermAdenomatous polyposis coli 423 TGG-TAG Trp-TermAdenomatous polyposis coli 424 CAG-TAG Gln-TermAdenomatous polyposis coli 433 CAG-TAG Gln-TermAdenomatous polyposis coli 443 GAA-TAA Glu-TermAdenomatous polyposis coli 457 TCA-TAA Ser-TermAdenomatous polyposis coli 473 CAG-TAG Gln-TermAdenomatous polyposis coli 486 TAC-TAG Tyr-TermAdenomatous polyposis coli 499 CGA-TGA Arg-TermAdenomatous polyposis coli 500 TAT-TAG Tyr-TermAdenomatous polyposis coli 541 CAG-TAG Gln-TermAdenomatous polyposis coli 553 TGG-TAG Trp-TermAdenomatous polyposis coli 554 CGA-TGA Arg-TermAdenomatous polyposis coli 564 CGA-TGA Arg-TermAdenomatous polyposis coli 577 TTA-TAA Leu-TermAdenomatous polyposis coli 586 AAA-TAA Lys-TermAdenomatous polyposis coli 592 TTA-TGA Leu-TermAdenomatous polyposis coli 593 TGG-TAG Trp-TermAdenomatous polyposis coli 593 TGG-TGA Trp-TermAdenomatous polyposis coli 622 TAC-TAA Tyr-TermAdenomatous polyposis coli 625 CAG-TAG Gln-TermAdenomatous polyposis coli 629 TTA-TAA Leu-TermAdenomatous polyposis coli 650 GAG-TAG Glu-TermAdenomatous polyposis coli 684 TTG-TAG Leu-TermAdenomatous polyposis coli 685 TGG-TGA Trp-TermAdenomatous polyposis coli 695 CAG-TAG Gln-TermAdenomatous polyposis coli 699 TGG-TGA Trp-TermAdenomatous polyposis coli 699 TGG-TAG Trp-TermAdenomatous polyposis coli 713 TCA-TGA Ser-TermAdenomatous polyposis coli 722 AGT-GGT Ser-GlyAdenomatous polyposis coli 747 TCA-TGA Ser-TermAdenomatous polyposis coli 764 TTA-TAA Leu-TermAdenomatous polyposis coli 784 TCT-ACT Ser-ThrAdenomatous polyposis coli 805 CGA-TGA Arg-TermAdenomatous polyposis coli 811 TCA-TGA Ser-TermAdenomatous polyposis coli 848 AAA-TAA Lys-TermAdenomatous polyposis coli 876 CGA-TGA Arg-TermAdenomatous polyposis coli 879 CAG-TAG Gln-TermAdenomatous polyposis coli 893 GAA-TAA Glu-TermAdenomatous polyposis coli 932 TCA-TAA Ser-TermAdenomatous polyposis coli 932 TCA-TGA Ser-TermAdenomatous polyposis coli 935 TAC-TAG Tyr-TermAdenomatous polyposis coli 935 TAC-TAA Tyr-TermAdenomatous polyposis coli 995 TGC-TGA Cys-TermAdenomatous polyposis coli 997 TAT-TAG Tyr-TermAdenomatous polyposis coli 999 CAA-TAA Gln-TermAdenomatous polyposis coli 1000 TAC-TAA Tyr-TermAdenomatous polyposis coli 1020 GAA-TAA Glu-TermAdenomatous polyposis coli 1032 TCA-TAA Ser-TermAdenomatous polyposis coli 1041 CAA-TAA Gln-TermAdenomatous polyposis coli 1044 TCA-TAA Ser-TermAdenomatous polyposis coli 1045 CAG-TAG Gln-TermAdenomatous polyposis coli 1049 TGG-TGA Trp-TermAdenomatous polyposis coli 1067 CAA-TAA Gln-TermAdenomatous polyposis coli 1071 CAA-TAA Gln-TermAdenomatous polyposis coli 1075 TAT-TAA Tyr-TermAdenomatous polyposis coli 1075 TAT-TAG Tyr-TermAdenomatous polyposis coli 1102 TAC-TAG Tyr-TermAdenomatous polyposis coli 1110 TCA-TGA Ser-TermAdenomatous polyposis coli 1114 CGA-TGA Arg-TermAdenomatous polyposis coli 1123 CAA-TAA Gln-TermAdenomatous polyposis coli 1135 TAT-TAG Tyr-TermAdenomatous polyposis coli 1152 CAG-TAG Gln-TermAdenomatous polyposis coli 1155 GAA-TAA Glu-TermAdenomatous polyposis coli 1168 GAA-TAA Glu-TermAdenomatous polyposis coli 1175 CAG-TAG Gln-TermAdenomatous polyposis coli 1176 CCT-CTT Pro-LeuAdenomatous polyposis coli 1184 GCC-CCC Ala-ProAdenomatous polyposis coli 1193 CAG-TAG Gln-TermAdenomatous polyposis coli 1194 TCA-TGA Ser-TermAdenomatous polyposis coli 1198 TCA-TGA Ser-TermAdenomatous polyposis coli 1201 TCA-TGA Ser-TermAdenomatous polyposis coli 1228 CAG-TAG Gln-TermAdenomatous polyposis coli 1230 CAG-TAG Gln-TermAdenomatous polyposis coli 1244 CAA-TAA Gln-TermAdenomatous polyposis coli 1249 TGC-TGA Cys-TermAdenomatous polyposis coli 1256 CAA-TAA Gln-TermAdenomatous polyposis coli 1262 TAT-TAA Tyr-TermAdenomatous polyposis coli 1270 TGT-TGA Cys-TermAdenomatous polyposis coli 1276 TCA-TGA Ser-TermAdenomatous polyposis coli 1278 TCA-TAA Ser-TermAdenomatous polyposis coli 1286 GAA-TAA Glu-TermAdenomatous polyposis coli 1289 TGT-TGA Cys-TermAdenomatous polyposis coli 1294 CAG-TAG Gln-TermAdenomatous polyposis coli 1307 ATA-AAA Ile-Lys Colorectal cancer,predisposition to, association 1309 GAA-TAA Glu-TermAdenomatous polyposis coli 1317 GAA-CAA Glu-Gln Colorectal cancer,predisposition to 1328 CAG-TAG Gln-Term Adenomatous polyposis coli 1338CAG-TAG Gln-Term Adenomatous polyposis coli 1342 TTA-TAA Leu-TermAdenomatous polyposis coli 1342 TTA-TGA Leu-TermAdenomatous polyposis coli 1348 AGG-TGG Arg-TrpAdenomatous polyposis coli 1357 GGA-TGA Gly-TermAdenomatous polyposis coli 1367 CAG-TAG Gln-TermAdenomatous polyposis coli 1370 AAA-TAA Lys-TermAdenomatous polyposis coli 1392 TCA-TAA Ser-TermAdenomatous polyposis coli 1392 TCA-TGA Ser-TermAdenomatous polyposis coli 1397 GAG-TAG Glu-TermAdenomatous polyposis coli 1449 AAG-TAG Lys-TermAdenomatous polyposis coli 1450 CGA-TGA Arg-TermAdenomatous polyposis coli 1451 GAA-TAA Glu-TermAdenomatous polyposis coli 1503 TCA-TAA Ser-TermAdenomatous polyposis coli 1517 CAG-TAG Gln-TermAdenomatous polyposis coli 1529 CAG-TAG Gln-TermAdenomatous polyposis coli 1539 TCA-TAA Ser-TermAdenomatous polyposis coli 1541 CAG-TAG Gln-TermAdenomatous polyposis coli 1564 TTA-TAA Leu-TermAdenomatous polyposis coli 1567 TCA-TGA Ser-TermAdenomatous polyposis coli 1640 CGG-TGG Arg-TrpAdenomatous polyposis coli 1693 GAA-TAA Glu-TermAdenomatous polyposis coli 1822 GAC-GTC Asp-Val Adenomatous polyposiscoli, association with ? 2038 CTG-GTG Leu-Val Adenomatous polyposis coli2040 CAG-TAG Gln-Term Adenomatous polyposis coli 2566 AGA-AAA Arg-LysAdenomatous polyposis coli 2621 TCT-TGT Ser-CysAdenomatous polyposis coli 2839 CTT-TTT Leu-PheAdenomatous polyposis coli

TABLE VIII NUCLEOTIDE SUBSTITUTIONS Relative Donor/Acceptor locationSubstitution Phenotype ds −1 G-C Adenomatous polyposis coli as −1 G-AAdenomatous polyposis coli as −1 G-C Adenomatous polyposis coli ds +2T-A Adenomatous polyposis coli as −1 G-C Adenomatous polyposis coli as−1 G-T Adenomatous polyposis coli as −1 G-A Adenomatous polyposis colias −2 A-C Adenomatous polyposis coli as −5 A-G Adenomatous polyposiscoli ds +3 A-C Adenomatous polyposis coli as −1 G-A Adenomatouspolyposis coli ds +1 G-A Adenomatous polyposis coli as −1 G-TAdenomatous polyposis coli ds +1 G-A Adenomatous polyposis coli as −1G-A Adenomatous polyposis coli ds +1 G-A Adenomatous polyposis coli ds+3 A-G Adenomatous polyposis coli ds +5 G-T Adenomatous polyposis colias −1 G-A Adenomatous polyposis coli as −6 A-G Adenomatous polyposiscoli as −5 A-G Adenomatous polyposis coli as −2 A-G Adenomatouspolyposis coli ds +2 T-C Adenomatous polyposis coli as −2 A-GAdenomatous polyposis coli ds +1 G-A Adenomatous polyposis coli ds +1G-T Adenomatous polyposis coli ds +2 T-G Adenomatous polyposis coli

TABLE IX APC SMALL DELETIONS Location/ codon Deletion Phenotype 77TTAgataGCAGTAATTT Adenomatous SEQ ID NO: 52 polyposis coli 97GGAAGccgggaagGATCTGTATC Adenomatous SEQ ID NO: 53 polyposis coli 138GAGAaAGAGAG_E3I3_GTAA Adenomatous SEQ ID NO: 54 polyposis coli 139AAAGAgag_E3I3_Gtaacttttct Thyroid cancer SEQ ID NO: 55 139AAAGagag_E3I3_GTAACTTTTC Adenomatous SEQ ID NO: 56 polyposis coli 142TTTTAAAAAAaAAAAATAG_I3E4_GTCA Adenomatous SEQ ID NO: 57 polyposis coli144 AAAATAG_I3E4_GTCatTGCTTCTTGC Adenomatous SEQ ID NO: 58polyposis coli 149 GACAaaGAAGAAAAGG Adenomatous SEQ ID NO: 59polyposis coli 149 GACAAagaaGAAAAGGAAA Adenomatous SEQ ID NO: 60polyposis coli 155 AGGAA{circumflex over ( )}AAAGActggtATTACGCTCAAdenomatous SEQ ID NO: 61 polyposis coli 169 AAAAGA{circumflex over( )}ATAGatagTCTTCCTTTA Adenomatous SEQ ID NO: 62 polyposis coli 172AGATAGT{circumflex over ( )}CTTcCTTTAACTGA Adenomatous SEQ ID NO: 63polyposis coli 179 TCCTTacaaACAGATATGA Adenomatous SEQ ID NO: 64polyposis coli 185 ACCaGAAGGCAATT Adenomatous SEQ ID NO: 65polyposis coli 196 ATCAGagTTGCGATGGA Adenomatous SEQ ID NO: 66polyposis coli 213 CGAGCaCAG_E5I5_GTAAGTT Adenomatous SEQ ID NO: 67polyposis coli 298 CACtcTGCACCTCGA Adenomatous SEQ ID NO: 68polyposis coli 329 GATaTGTCGCGAAC Adenomatous SEQ ID NO: 69polyposis coli 365 AAAGActCTGTATTGTT Adenomatous SEQ ID NO: 70polyposis coli 397 GACaaGAGAGGCAGG Adenomatous SEQ ID NO: 71polyposis coli 427 CATGAacCAGGCATGGA Adenomatous SEQ ID NO: 72polyposis coli 428 GAACCaGGCATGGACC Adenomatous SEQ ID NO: 73polyposis coli 436 AATCCaa_E9I9_gTATGTTCTCT Adenomatous SEQ ID NO: 74polyposis coli 440 GCTCCtGTTGAACATC Adenomatous SEQ ID NO: 75polyposis coli 455 AAACTtTCATTTGATG Adenomatous SEQ ID NO: 76polyposis coli 455 AAACtttcaTTTGATGAAG Adenomatous SEQ ID NO: 77polyposis coli 472 CTAcAGGCCATTGC Adenomatous SEQ ID NO: 78polyposis coli 472 TAAATTAG_I10E11_GGgGACTACAGGC AdenomatousSEQ ID NO: 79 polyposis coli 478 TTATtGCAAGTGGAC AdenomatousSEQ ID NO: 80 polyposis coli 486 TACGgGCTTACTAAT AdenomatousSEQ ID NO: 81 polyposis coli 494 AGTATtACACTAAGAC AdenomatousSEQ ID NO: 82 polyposis coli 495 ATTACacTAAGACGATA AdenomatousSEQ ID NO: 83 polyposis coli 497 CTAaGACGATATGC AdenomatousSEQ ID NO: 84 polyposis coli 520 TGCTCtaTGAAAGGCTG AdenomatousSEQ ID NO: 85 polyposis coli 526 ATGAGagcacttgtgGCCCAACTAA AdenomatousSEQ ID NO: 86 polyposis coli 539 GACTTaCAGCAG_E12I12_GTAC AdenomatousSEQ ID NO: 87 polyposis coli 560 AAAAAgaCGTTGCGAGA AdenomatousSEQ ID NO: 88 polyposis coli 566 GTTGgaagtGTGAAAGCAT AdenomatousSEQ ID NO: 89 polyposis coli 570 AAAGCaTTGATGGAAT AdenomatousSEQ ID NO: 90 polyposis coli 577 TTAGaagtTAAAAAG_E13I13_GTA AdenomatousSEQ ID NO: 91 polyposis coli 584 ACCCTcAAAAGCGTAT AdenomatousSEQ ID NO: 92 polyposis coli 591 GCCTtATGGAATTTG AdenomatousSEQ ID NO: 93 polyposis coli 608 GCTgTAGATGGTGC AdenomatousSEQ ID NO: 94 polyposis coli 617 GTTggcactcttacttaccGGAGCCAGACAdenomatous SEQ ID NO: 95 polyposis coli 620 CTTACttacCGGAGCCAGAAdenomatous SEQ ID NO: 96 polyposis coli 621 ACTTaCCGGAGCCAG AdenomatousSEQ ID NO: 97 polyposis coli 624 AGCcaGACAAACACT AdenomatousSEQ ID NO: 98 polyposis coli 624 AGCCagacAAACACTTTA AdenomatousSEQ ID NO: 99 polyposis coli 626 ACAaacaCTTTAGCCAT AdenomatousSEQ ID NO: 100 polyposis coli 629 TTAGCcATTATTGAAA AdenomatousSEQ ID NO: 101 polyposis coli 635 GGAGgTGGGATATTA AdenomatousSEQ ID NO: 102 polyposis coli 638 ATATtACGGAATGTG AdenomatousSEQ ID NO: 103 polyposis coli 639 TTACGgAATGTGTCCA AdenomatousSEQ ID NO: 104 polyposis coli 657 AGAgaGAACAACTGT AdenomatousSEQ ID NO: 105 polyposis coli 659 TATTTCAG_I14E15_GCaaatcctaagagagAACAAAdenomatous SEQ ID NO: 106 CTGTC polyposis coli 660 AACTgtCTACAAACTTAdenomatous SEQ ID NO: 107 polyposis coli 665 TTAttACAACACTTAAdenomatous SEQ ID NO: 108 polyposis coli 668 CACttAAAATCTCATAdenomatous SEQ ID NO: 109 polyposis coli 673 AGTttgacaatagtCAGTAATGCAAdenomatous SEQ ID NO: 110 polyposis coli 768 CACTTaTCAGAAACTTAdenomatous SEQ ID NO: 111 polyposis coli 769 TTATcAGAAACTTTTAdenomatous SEQ ID NO: 112 polyposis coli 770 TCAGAaACTTTTGACAAdenomatous SEQ ID NO: 113 polyposis coli 780 AGTCcCAAGGCATCTAdenomatous SEQ ID NO: 114 polyposis coli 792 AAGCaAAGTCTCTATAdenomatous SEQ ID NO: 115 polyposis coli 792 AAGCAaaGTCTCTATGGAdenomatous SEQ ID NO: 116 polyposis coli 793 CAAAgTCTCTATGGTAdenomatous SEQ ID NO: 117 polyposis coli 798 GATTatGTTTTTGACAAdenomatous SEQ ID NO: 118 polyposis coli 802 GACACcaatcgacatGATGATAATAAdenomatous SEQ ID NO: 119 polyposis coli 805 CGACatGATGATAATAAdenomatous SEQ ID NO: 120 polyposis coli 811 TCAGacaaTTTTAATACTAdenomatous SEQ ID NO: 121 polyposis coli 825 TATtTGAATACTAC AdenomatousSEQ ID NO: 122 polyposis coli 827 AATAcTACAGTGTTA AdenomatousSEQ ID NO: 123 polyposis coli 830 GTGTTacccagctcctctTCATCAAGAGAdenomatous SEQ ID NO: 124 polyposis coli 833 AGCTCcTCTTCATCAAAdenomatous SEQ ID NO: 125 polyposis coli 836 TCATcAAGAGGAAGCAdenomatous SEQ ID NO: 126 polyposis coli 848 AAAGAtaGAAGTTTGGAAdenomatous SEQ ID NO: 127 polyposis coli 848 AAAGatagaagTTTGGAGAGAAdenomatous SEQ ID NO: 128 polyposis coli 855 GAACgCGGAATTGGTAdenomatous SEQ ID NO: 129 polyposis coli 856 CGCGgaattGGTCTAGGCAAdenomatous SEQ ID NO: 130 polyposis coli 856 CGCGgAATTGGTCTAAdenomatous SEQ ID NO: 131 polyposis coli 879 CAGaTCTCCACCAC AdenomatousSEQ ID NO: 132 polyposis coli 902 GAAGAcagaAGTTCTGGGT AdenomatousSEQ ID NO: 133 polyposis coli 907 GGGTcTACCACTGAA AdenomatousSEQ ID NO: 134 polyposis coli 915 GTGACaGATGAGAGAA AdenomatousSEQ ID NO: 135 polyposis coli 929 CATACacatTCAAACACTT AdenomatousSEQ ID NO: 136 polyposis coli 930 ACACAttcaAACACTTACA AdenomatousSEQ ID NO: 137 polyposis coli 931 CATtCAAACACTTA AdenomatousSEQ ID NO: 138 polyposis coli 931 CATTcAAACACTTAC AdenomatousSEQ ID NO: 139 polyposis coli 933 AACacttACAATTTCAC AdenomatousSEQ ID NO: 140 polyposis coli 935 TACAatttcactAAGTCGGAAA AdenomatousSEQ ID NO: 141 polyposis coli 937 TTCActaaGTCGGAAAAT AdenomatousSEQ ID NO: 142 polyposis coli 939 AAGtcggAAAATTCAAA AdenomatousSEQ ID NO: 143 polyposis coli 946 ACATgTTCTATGCCT AdenomatousSEQ ID NO: 144 polyposis coli 954 TTAGaaTACAAGAGAT AdenomatousSEQ ID NO: 145 polyposis coli 961 AATgATAGTTTAAA AdenomatousSEQ ID NO: 146 polyposis coli 963 AGTTTaAATAGTGTCA AdenomatousSEQ ID NO: 147 polyposis coli 964 TTAaataGTGTCAGTAG AdenomatousSEQ ID NO: 148 polyposis coli 973 TATGgTAAAAGAGGT AdenomatousSEQ ID NO: 149 polyposis coli 974 GGTAAaAGAGGTCAAA AdenomatousSEQ ID NO: 150 polyposis coli 975 AAAAgaGGTCAAATGA Thyroid cancerSEQ ID NO: 151 992 AGTAAgTTTTGCAGTT Thyroid cancer SEQ ID NO: 152 993AAGttttgcagttaTGGTCAATAC Adenomatous SEQ ID NO: 153 polyposis coli 999CAAtacccagCCGACCTAGC Adenomatous SEQ ID NO: 154 polyposis coli 1023ACACcAATAAATTAT Adenomatous SEQ ID NO: 155 polyposis coli 1030AAAtATTCAGATGA Adenomatous SEQ ID NO: 156 polyposis coli 1032TCAGatgagCAGTTGAACT Adenomatous SEQ ID NO: 157 polyposis coli 1033GATGaGCAGTTGAAC Adenomatous SEQ ID NO: 158 polyposis coli 1049TGGGcAAGACCCAAA Adenomatous SEQ ID NO: 159 polyposis coli 1054CACAtaataGAAGATGAAA Adenomatous SEQ ID NO: 160 polyposis coli 1055ATAAtagaaGATGAAATAA Adenomatous SEQ ID NO: 161 polyposis coli 1056ATAGAaGATGAAATAA Adenomatous SEQ ID NO: 162 polyposis coli 1060ATAAAacaaaGTGAGCAAAG Adenomatous SEQ ID NO: 163 polyposis coli 1061AAAcaaaGTGAGCAAAG Adenomatous SEQ ID NO: 164 polyposis coli 1061AAACaaAGTGAGCAAA Adenomatous SEQ ID NO: 165 polyposis coli 1062CAAAgtgaGCAAAGACAA Adenomatous SEQ ID NO: 166 polyposis coli 1065CAAAGacAATCAAGGAA Adenomatous SEQ ID NO: 167 polyposis coli 1067CAAtcaaGGAATCAAAG Adenomatous SEQ ID NO: 168 polyposis coli 1071CAAAgtACAACTTATC Adenomatous SEQ ID NO: 169 polyposis coli 1079ACTGagAGCACTGATG Adenomatous SEQ ID NO: 170 polyposis coli 1082ACTGAtgATAAACACCT Adenomatous SEQ ID NO: 171 polyposis coli 1084GATaaacACCTCAAGTT Adenomatous SEQ ID NO: 172 polyposis coli 1086CACCtcAAGTTCCAAC Adenomatous SEQ ID NO: 173 polyposis coli 1093TTTGgACAGCAGGAA Adenomatous SEQ ID NO: 174 polyposis coli 1098TGTgtTTCTCCATAC Adenomatous SEQ ID NO: 175 polyposis coli 1105CGGgGAGCCAATGG Thyroid cancer SEQ ID NO: 176 1110 TCAGAaACAAATCGAGAdenomatous SEQ ID NO: 177 polyposis coli 1121 ATTAAtcaaAATGTAAGCCAdenomatous SEQ ID NO: 178 polyposis coli 1131 CAAgAAGATGACTAAdenomatous SEQ ID NO: 179 polyposis coli 1134 GACTAtGAAGATGATAAdenomatous SEQ ID NO: 180 polyposis coli 1137 GATgataaGCCTACCAATAdenomatous SEQ ID NO: 181 polyposis coli 1146 CGTTAcTCTGAAGAAGAdenomatous SEQ ID NO: 182 polyposis coli 1154 GAAGaagaaGAGAGACCAAAdenomatous SEQ ID NO: 183 polyposis coli 1155 GAAGaagaGAGACCAACAAdenomatous SEQ ID NO: 184 polyposis coli 1156 GAAgagaGACCAACAAAAdenomatous SEQ ID NO: 185 polyposis coli 1168 GAAgagaaACGTCATGTGAdenomatous SEQ ID NO: 186 polyposis coli 1178 GATTAtagtttaAAATATGCCAAdenomatous SEQ ID NO: 187 polyposis coli 1181 TTAAaATATGCCACAAdenomatous SEQ ID NO: 188 polyposis coli 1184 GCCacagaTATTCCTTCAAdenomatous SEQ ID NO: 189 polyposis coli 1185 ACAgaTATTCCTTCAAdenomatous SEQ ID NO: 190 polyposis coli 1190 TCACAgAAACAGTCATAdenomatous SEQ ID NO: 191 polyposis coli 1192 AAAcaGTCATTTTCAAdenomatous SEQ ID NO: 192 polyposis coli 1198 TCAaaGAGTTCATCTAdenomatous SEQ ID NO: 193 polyposis coli 1207 AAAAcCGAACATATGAdenomatous SEQ ID NO: 194 polyposis coli 1208 ACCgaacATATGTCTTCAdenomatous SEQ ID NO: 195 polyposis coli 1210 CATatGTCTTCAAGCAdenomatous SEQ ID NO: 196 polyposis coli 1233 CCAAGtTCTGCACAGAAdenomatous SEQ ID NO: 197 polyposis coli 1249 TGCAaaGTTTCTTCTAAdenomatous SEQ ID NO: 198 polyposis coli 1259 ATAcaGACTTATTGTAdenomatous SEQ ID NO: 199 polyposis coli 1260 CAGACttATTGTGTAGAAdenomatous SEQ ID NO: 200 polyposis coli 1268 CCAaTATGTTTTTCAdenomatous SEQ ID NO: 201 polyposis coli 1275 AGTtCATTATCATCAdenomatous SEQ ID NO: 202 polyposis coli 1294 CAGGAaGCAGATTCTGAdenomatous SEQ ID NO: 203 polyposis coli 1301 ACCCtGCAAATAGCAAdenomatous SEQ ID NO: 204 polyposis coli 1306 GAAAtaaaAGAAAAGATTAdenomatous SEQ ID NO: 205 polyposis coli 1307 ATAaAAGAAAAGATAdenomatous SEQ ID NO: 206 polyposis coli 1308 AAAgaaaAGATTGGAACAdenomatous SEQ ID NO: 207 polyposis coli 1308 AAAGAaaagaTTGGAACTAGAdenomatous SEQ ID NO: 208 polyposis coli 1318 GATCcTGTGAGCGAAAdenomatous SEQ ID NO: 209 polyposis coli 1320 GTGAGcGAAGTTCCAGAdenomatous SEQ ID NO: 210 polyposis coli 1323 GTTCcAGCAGTGTCAAdenomatous SEQ ID NO: 211 polyposis coli 1329 CACCctagaaccAAATCCAGCAAdenomatous SEQ ID NO: 212 polyposis coli 1336 AGACtgCAGGGTTCTAAdenomatous SEQ ID NO: 213 polyposis coli 1338 CAGgGTTCTAGTTTAdenomatous SEQ ID NO: 214 polyposis coli 1340 TCTAgTTTATCTTCAAdenomatous SEQ ID NO: 215 polyposis coli 1342 TTATcTTCAGAATCAAdenomatous SEQ ID NO: 216 polyposis coli 1352 GTTgAATTTTCTTCAdenomatous SEQ ID NO: 217 polyposis coli 1361 CCCTcCAAAAGTGGTAdenomatous SEQ ID NO: 218 polyposis coli 1364 AGTggtgCTCAGACACCAdenomatous SEQ ID NO: 219 polyposis coli 1371 AGTCCacCTGAACACTAAdenomatous SEQ ID NO: 220 polyposis coli 1372 CCACCtGAACACTATGAdenomatous SEQ ID NO: 221 polyposis coli 1376 TATGttCAGGAGACCCAdenomatous SEQ ID NO: 222 polyposis coli 1394 GATAgtTTTGAGAGTCAdenomatous SEQ ID NO: 223 polyposis coli 1401 ATTGCcAGCTCCGTTCAdenomatous SEQ ID NO: 224 polyposis coli 1415 AGTGGcATTATAAGCCAdenomatous SEQ ID NO: 225 polyposis coli 1426 AGCCcTGGACAAACCAdenomatous SEQ ID NO: 226 polyposis coli 1427 CCTGGaCAAACCATGCAdenomatous SEQ ID NO: 227 polyposis coli 1431 ATGCcACCAAGCAGAAdenomatous SEQ ID NO: 228 polyposis coli 1454 AAAAAtAAAGCACCTAAdenomatous SEQ ID NO: 229 polyposis coli 1461 GAAaAGAGAGAGAGAdenomatous SEQ ID NO: 230 polyposis coli 1463 AGAgagaGTGGACCTAAAdenomatous SEQ ID NO: 231 polyposis coli 1464 GAGAgTGGACCTAAGAdenomatous SEQ ID NO: 232 polyposis coli 1464 GAGAgtGGACCTAAGCAdenomatous SEQ ID NO: 233 polyposis coli 1464 GAGagTGGACCTAAGAdenomatous SEQ ID NO: 234 polyposis coli 1492 GCCaCGGAAAGTACAdenomatous SEQ ID NO: 235 polyposis coli 1493 ACGGAaAGTACTCCAGAdenomatous SEQ ID NO: 236 polyposis coli 1497 CCAgATGGATTTTCAdenomatous SEQ ID NO: 237 polyposis coli 1503 TCAtccaGCCTGAGTGCAdenomatous SEQ ID NO: 238 polyposis coli 1522 TTAagaataaTGCCTCCAGTAdenomatous SEQ ID NO: 239 polyposis coli 1536 GAAACagAATCAGAGCAAdenomatous SEQ ID NO: 240 polyposis coli 1545 TCAAAtgaaaACCAAGAGAAAdenomatous SEQ ID NO: 241 polyposis coli 1547 GAAaACCAAGAGAAAdenomatous SEQ ID NO: 242 polyposis coli 1550 GAGAaagaGGCAGAAAAAAdenomatous SEQ ID NO: 243 polyposis coli 1577 GAATgtATTATTTCTGAdenomatous SEQ ID NO: 244 polyposis coli 1594 CCAGCcCAGACTGCTTAdenomatous SEQ ID NO: 245 polyposis coli 1596 CAGACtGCTTCAAAATAdenomatous SEQ ID NO: 246 polyposis coli 1823 TTCAaTGATAAGCTCAdenomatous SEQ ID NO: 247 polyposis coli 1859 AATGAttctTTGAGTTCTCAdenomatous SEQ ID NO: 248 polyposis coli 1941 CCAGAcagaGGGGCAGCAADesmoid SEQ ID NO: 249 tumours 1957 GAAaATACTCCAGT AdenomatousSEQ ID NO: 250 polyposis coli 1980 AACaATAAAGAAAA AdenomatousSEQ ID NO: 251 polyposis coli 1985 GAACCtATCAAAGAGA AdenomatousSEQ ID NO: 252 polyposis coli 1986 CCTaTCAAAGAGAC AdenomatousSEQ ID NO: 253 polyposis coli 1998 GAACcAAGTAAACCT AdenomatousSEQ ID NO: 254 polyposis coli 2044 AGCTCcGCAATGCCAA AdenomatousSEQ ID NO: 255 polyposis coli 2556 TCATCccttcctcGAGTAAGCAC AdenomatousSEQ ID NO: 256 polyposis coli 2643 CTAATttatCAAATGGCAC AdenomatousSEQ ID NO: 257 polyposis coli

Bold letters indicate the codon. Undercase letters represent thedeletion. Where deletions extend beyond the coding region, otherpositional information is provided. For example, the abbreviation 5′ UTRrepresents 5′ untranslated region, and the abbreviation E6I6 denotesexon 6/intron 6 boundary.

TABLE X SMALL INSERTIONS Codon Insertion Phenotype 157 TAdenomatous polyposis coli 170 AGAT Adenomatous polyposis coli 172 TAdenomatous polyposis coli 199 G Adenomatous polyposis coli 243 AGAdenomatous polyposis coli 266 T Adenomatous polyposis coli 357 AAdenomatous polyposis coli 405 C Adenomatous polyposis coli 413 TAdenomatous polyposis coli 416 A Adenomatous polyposis coli 457 GAdenomatous polyposis coli 473 A Adenomatous polyposis coli 503 ATTCAdenomatous polyposis coli 519 C Adenomatous polyposis coli 528 AAdenomatous polyposis coli 561 A Adenomatous polyposis coli 608 AAdenomatous polyposis coli 620 CT Adenomatous polyposis coli 621 AAdenomatous polyposis coli 623 TTAC Adenomatous polyposis coli 627 AAdenomatous polyposis coli 629 A Adenomatous polyposis coli 636 GTAdenomatous polyposis coli 639 A Adenomatous polyposis coli 704 TAdenomatous polyposis coli 740 ATGC Adenomatous polyposis coli 764 TAdenomatous polyposis coli 779 TT Adenomatous polyposis coli 807 ATAdenomatous polyposis coli 827 AT Adenomatous polyposis coli 831 AAdenomatous polyposis coli 841 CTTA Adenomatous polyposis coli 865 CTAdenomatous polyposis coli 865 AT Adenomatous polyposis coli 900 TGAdenomatous polyposis coli 921 G Adenomatous polyposis coli 927 AAdenomatous polyposis coli 935 A Adenomatous polyposis coli 936 CAdenomatous polyposis coli 975 A Adenomatous polyposis coli 985 TAdenomatous polyposis coli 997 A Adenomatous polyposis coli 1010 TAAdenomatous polyposis coli 1085 C Adenomatous polyposis coli 1085 ATAdenomatous polyposis coli 1095 A Adenomatous polyposis coli 1100 GTTTAdenomatous polyposis coli 1107 GGAG Adenomatous polyposis coli 1120 GAdenomatous polyposis coli 1166 A Adenomatous polyposis coli 1179 TAdenomatous polyposis coli 1187 A Adenomatous polyposis coli 1211 TAdenomatous polyposis coli 1256 A Adenomatous polyposis coli 1265 TAdenomatous polyposis coli 1267 GATA Adenomatous polyposis coli 1268 TAdenomatous polyposis coli 1301 A Adenomatous polyposis coli 1301 CAdenomatous polyposis coli 1323 A Adenomatous polyposis coli 1342 TAdenomatous 

posis coli 1382 T Adenomatous polyposis coli 1458 GTAGAdenomatous polyposis coli 1463 AG Adenomatous polyposis coli 1488 TAdenomatous polyposis coli 1531 A Adenomatous polyposis coli 1533 TAdenomatous polyposis coli 1554 A Adenomatous polyposis coli 1555 AAdenomatous polyposis coli 1556 T Adenomatous polyposis coli 1563 GACCTAdenomatous polyposis coli 1924 AA Desmoid tumours

indicates data missing or illegible when filed

TABLE XI SMALL INSERTIONS/DELETIONS Location/ codon Deletion InsertionPhenotype 538 GAAGAcTTACAGCAGG gaa Adenomatous SEQ ID NO: 258polyposis coli 620 CTTACttaCCGGAGCCAG ct Adenomatous SEQ ED NO: 259polyposis coli 728 AATctcatGGCAAATAGG ttgcagctttaa Adenomatous(SEQ ID NO: 260) (SEQ ID NO: polyposis coli 261) 971 GATGgtTATGGTAAAAtaa Adenomatous SEQ ID NO: 262 polyposis coli

TABLE XII GROSS DELETIONS 2 kb including ex. 11 Adenomatous polyposiscoli 3 kb I10E11 − 1.5 kb to I12E13 − 170 bp Adenomatous polyposis coli335 bp nt. 1409-1743 ex. 11-13 Adenomatous polyposis coli 6 kb incl. ex.14 Adenomatous polyposis coli 817 bp I13E14 − 679 to I13E14 + 138Adenomatous polyposis coli ex. 11-15M Adenomatous polyposis coli ex.11-3′UTR Adenomatous polyposis coli ex. 15A-ex. 15F Adenomatouspolyposis coli ex. 4 Adenomatous polyposis coli ex. 7, 8 and 9Adenomatous polyposis coli ex. 8 to beyond ex. 15F Adenomatous polyposiscoli ex. 8-ex. 15F Adenomatous polyposis coli ex. 9 Adenomatouspolyposis coli >10mb (del 5q22) Adenomatous polyposis coli

TABLE XIII GROSS INSERTIONS AND DUPLICATIONS Description PhenotypeInsertion of 14 bp nt. 3816 Adenomatous polyposis coli Insertion of 22bp nt. 4022 Adenomatous polyposis coli Duplication of 43 bp cd. 1295Adenomatous polyposis coli Insertion of 337 bp of Alu I sequence cd.Desmoid tumours 1526

TABLE XIV COMPLEX REARRANGEMENTS (INCLUDING INVERSIONS) A-T nt. 4893Q1625H, Del C nt. 4897 cd. Adenomatous polyposis coli 1627 Del 1099 bpI13E14 − 728 to E14I14 + Adenomatous polyposis coli 156, ins 126 bp Del1601 bp E14I14 + 27 to E14I14 + Adenomatous polyposis coli 1627, ins 180bp Del 310 bp, ins. 15 bp nt. 4394, cd 1464 Adenomatous polyposis coliDel A and T cd. 1395 Adenomatous polyposis coli Del TC nt. 4145, Del TGTnt. 4148 Adenomatous polyposis coli Del. T, nt. 983, Del. 70 bp, nt. 985Adenomatous polyposis coli Del. nt. 3892-3903, ins ATTT Adenomatouspolyposis coli

TABLE XV DIAGNOSTIC APPLICATIONS Cancer Type Marker ApplicationReference Breast Her2/Neu Detection - Using methods described herein,design D. Xie et al., J. Natl. polymorphism at second primer such thatafter PCR, and Cancer Institute, 92, codon 655 digestion withrestriction enzyme, a 5′ 412 (2000) (GTC/valine to overhang containingDNA sequence for K. S. Wilson et al., ATC/isoleucine codon 655 ofHer2/Neu is generated. Am. J. [Val(655)Ile]) Her2/Neu can be detectedand Pathol., 161, 1171 quantified as a possible marker for (2002) breastcancer. Methods described L. Newman, Cancer herein can detect bothmutant allele and Control, 9, 473 (2002) normal allele, even when mutantallele is small fraction of total DNA. Herceptin therapy for breastcancer is based upon screening for Her2. The earlier the mutant allelecan be detected, the faster therapy can be provided. Breast/OvarianHypermethylation Methods described herein can be used M. Esteller etal., of BRCA1 to differentiate between tumors New England Jnl resultingfrom inherited BRCA1 Med., 344, 539 mutations and those fromnon-inherited (2001) abnormal methylation of the gene BladderMicrosatellite Methods described herein can be W. G. Bas et al.,analysis of free applied to microsatellite analysis and Clinical Cancertumor DNA in FGFR3 mutation analysis for detection Res., 9, 257 (2003)Urine, Serum and of bladder cancer. Methods described M. Utting et al.,Plasma herein provide a non-invasive method Clinical Cancer Res., fordetection of bladder cancer. 8, 35 (2002) L. Mao, D. Sidransky et al.,Science, 271, 669 (1996) Lung Microsatellite Methods described hereincan be used T. Liloglou et al., analysis of DNA to detect mutations insputum samples, Cancer Research, 61, from sputum and can markedly boostthe accuracy of 1624, (2001) preclinical lung cancer screening M.Tockman et al., Cancer Control, 7, 19 (2000) Field et al., CancerResearch, 59, 2690 (1999) Cervical Analysis of HPV Methods describedherein can be used N. Munoz et al., New genotype to detect HPV genotypefrom a cervical England Jnl Med., smear preparation. 348, 518 (2003)Head and Neck Tumor specific Methods described herein can be used M.Spafford et al. alterations in to detect any of 23 microsatelliteClinical Cancer exfoliated oral markers, which are associated withResearch, 17, 607 mucosal cells Head and Neck Squamous Cell (2001)(microsatellite Carcinoma (HNSCC). A. El-Naggar et al., J. markers) Mol.Diag., 3, 164 (2001) Colorectal Screening for Methods described hereincan be used B. Ryan et al. mutation in K-ras2 to detect K-ras 2mutations, which can Gut, 52, 101 (2003) and APC genes. be used as aprognostic indicator for colorectal cancer. APC (see Example 5).Prostate GSTP1 Methods described herein can be used P. Cairns et al.Clin. Hypermethylation to detect GSTP1 hypermethylation in Can. Res., 7,2727 urine from patients with prostate cancer; (2001) this can be a moreaccurate indicator than PSA.

HIV Antiretroviral Screening Methods described herein can be used J.Durant et al. resistance individuals for for detection of mutations inthe HIV The Lancet, 353, mutations in HIV virus. Treatment outcomes are2195 (1999) virus - e.g. 154V improved in individuals receiving mutationor CCR5 anti-retroviral therapy based upon Δ 32 allele. resistancescreening.

Cardiology Congestive Synergistic Methods described herein can be usedK. Small et al. New Heart Failure polymorphisms of to genotype theseloci and may help Eng. Jnl. Med., beta1 and alpha2c identify people whoare at a higher 347, 1135 (2002) adrenergic risk of heart failure.receptors

Example 8

Single nucleotide polymorphisms (SNPs) represent the most common form ofsequence variation; three million common SNPs with a populationfrequency of over 5% have been estimated to be present in the humangenome. A genetic map using these polymorphisms as a guide is beingdeveloped (http://research.marshfieldclinic.org/genetics/; internetaddress as of Feb. 13, 2003).

The allele frequency varies from SNP to SNP; the allele frequency forone SNP may be 50:50, while the allele frequency for another SNP may be90:10. The closer the allele frequency is to 50:50, the more likely anyparticular individual will be heterozygous at that SNP. The SNPconsortium provides allele frequency information for some SNPs but notfor others. www.snp.chsl.org. The allele frequency for a particular SNPprovides valuable information as to the utility of that SNP for thenon-invasive prenatal screening method described in Example 5. While allSNPs can be used, SNPs with allele frequencies closer to 50:50 arepreferable.

Briefly, maternal blood contains fetal DNA. Maternal DNA can bedistinguished from fetal DNA by examining SNPs wherein the mother ishomozygous. For example, at SNP X, the maternal DNA may be homozygousfor guanine. If template DNA obtained from the plasma of a pregnantfemale is heterozygous, as demonstrated by the detection of signalscorresponding to an adenine allele and an guanine allele, the adenineallele can be used as a beacon for the fetal DNA (see Example 5). Thecloser the allele frequency of a SNP is to 50:50, the more likely therewill be allele differences at a particular SNP between the maternal DNAand the fetal DNA.

For example, if at SNP X the observed alleles are adenine and guanine,and the SNP has an allele frequency of 90(A):10(G), it is likely thatboth mother and father will be homozygous for adenine at that particularSNP. Thus, both the maternal DNA and the fetal DNA will be homozygousfor adenine, and there is no distinct signal for the fetal DNA. However,if at SNP X the allele frequency is 50:50, and the mother is homozygousfor adenine, the probability is higher that the paternal DNA willcontain a guanine allele at SNP X.

Below, a method for determining the allele frequency for a SNP isprovided. Seven SNPs located on chromosome 13 were analyzed. The methodis applicable for any SNP including but not limited to the SNPs on humanchromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, X and Y.

Preparation of Template DNA

To determine the allele frequency of a particular SNP, DNA was obtainedfrom two hundred and fifty individuals after informed consent had beengranted. From each individual, a 9 ml blood sample was collected into asterile tube (Fischer Scientific, 9 ml EDTA Vacuette tubes, catalognumber NC9897284). The tubes were spun at 1000 rpm for ten minutes. Thesupernatant (the plasma) of each sample was removed, and one milliliterof the remaining blood sample, which is commonly referred to as the“buffy-coat” was transferred to a new tube. One milliliter of 1×PBS wasadded to each sample.

Template DNA was isolated using the QIAmp DNA Blood Midi Kit supplied byQIAGEN (Catalog number 51183). The template DNA was isolated as perinstructions included in the kit. From each individual, 0.76 μg of DNAwas pooled together, and the pooled DNA was used in all subsequentreactions.

Design of Primers

SNP TSC0903430 was amplified using the following primer set:

First primer: (SEQ ID NO: 279) 5′GTCTTGCATGTAGAATTCTAGGGACGCTGCTTTTCGTC 3′ Second primer:(SEQ ID NO: 280) 5′ CTCCTAGACATCGGGACTAGAATGTCCAC 3′

The first primer contained a recognition site for the restriction enzymeEcoRI, and was designed to anneal eighty-two bases from the locus ofinterest. The second primer contained the recognition site for therestriction enzyme BsmF I.

SNP TSC0337961 was amplified using the following primer set:

First primer: (SEQ D NO: 281) 5′ACACAAGGCAGAGAATTCCAGTCCTGAGGGTGGGGGCC 3′ Second primer:(SEQ ID NO: 282) 5′ CCGTGTTTTAACGGGACAAGCTGTTCTTC 3′

The first primer contained a recognition site for the restriction enzymeEcoRI, and was designed to anneal ninety-two bases from the locus ofinterest. The second primer contained the recognition site for therestriction enzyme BsmF I.

SNP TSC0786441 was amplified using the following primer set:

First primer: (SEQ ID NO: 283) 5′GTAGCGGAGGTTGAATTCTATATGTTGTCTTGGACATT 3′ Second primer:(SEQ ID NO: 284) 5′ CATCAGTAGAGTGGGACGAAAGTTCTGGC 3′

The first primer contained a recognition site for the restriction enzymeEcoRI, and was designed to anneal one hundred and four bases from thelocus of interest. The second primer contained the recognition site forthe restriction enzyme BsmF I.

SNP TSC1168303 was amplified using the following primer set:

First primer: (SEQ ID NO: 285) 5′ATCCACGCCGCAGAATTCGTATTCATGGGCATGTCAAA 3′ Second primer:(SEQ ID NO: 286) 5′ CTTGGGACTATTGGGACCAGTGTTCAATC 3′

The first primer contained a recognition site for the restriction enzymeEcoRI, and was designed to anneal sixty-four bases from the locus ofinterest. The second primer contained the recognition site for therestriction enzyme BsmF I.

SNP TSC0056188 was amplified using the following primer set:

First primer: (SEQ ID NO: 287) 5′CCAGAAAGCCGTGAATTCGTTAAGCCAACCTGACTCCA 3′ Second primer:(SEQ ID NO: 288) 5′ TCGGGGTTAGTCGGGACATCCAGCAGCCC 3′

The first primer contained a recognition site for the restriction enzymeEcoRI, and was designed to anneal eighty-two bases from the locus ofinterest. The second primer contained the recognition site for therestriction enzyme BsmF I.

SNP TSC0466177 was amplified using the following primer s

First primer: (SEQ ID NO: 289) 5′CGAAGGTAATGTGAATTCCAAAACTTAGTGCCACAATT 3′ Second primer:(SEQ ID NO: 290) 5′ ATACCGCCCAACGGGACAGATCCATTGAC 3′

The first primer contained a recognition site for the restriction enzymeEcoRI, and was designed to anneal ninety-two bases from the locus ofinterest. The second primer contained the recognition site for therestriction enzyme BsmF I.

SNP TSC0197424 was amplified using the following primer set:

First primer: (SEQ ID NO: 291) 5′AGAAACCTGTAAGAATTCGATTCCAAATTGTTTTTTGG 3′ Second primer:(SEQ ID NO: 292) 5′ CGATCATAGGGGGGGACAGGAGAGAGCAC 3′

The first primer contained a recognition site for the restriction enzymeEcoRI, and was designed to anneal one hundred and four bases from thelocus of interest. The second primer contained the recognition site forthe restriction enzyme BsmF I.

The first primer was designed to anneal at various distances from thelocus of interest. The skilled artisan understands that the annealinglocation of the first primer can be any distance from the locus ofinterest including but not limited to 5-10, 11-15, 16-20, 21-25, 26-30,31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80,81-85, 86-90, 91-95, 96-100, 101-105, 106-110, 111-115, 116-120,121-125, 126-130, 131-140, 141-160, 161-180, 181-200, 201-220, 221-240,241-260, 261-280, 281-300, 301-350, 351-400, 401-450, 451-500, 501-1000,1001-2000, 2001-3000, or greater than 3000.

All loci of interest were amplified from the template genomic DNA usingthe polymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and4,683,202, incorporated herein by reference). In this example, the lociof interest were amplified in separate reaction tubes but they can alsobe amplified together in a single PCR reaction. For increasedspecificity, a “hot-start” PCR was used. PCR reactions were performedusing the HotStarTaq Master Mix Kit supplied by QIAGEN (catalog number203443). The amount of template DNA and primer per reaction can beoptimized for each locus of interest. In this example, 40 ng of templatehuman genomic DNA (a mixture of template DNA from 245 individuals) and 5μM of each primer were used. Forty cycles of PCR were performed. Thefollowing PCR conditions were used:

-   -   (1) 95° C. for 15 minutes and 15 seconds;    -   (2) 37° C. for 30 seconds;    -   (3) 95° C. for 30 seconds;    -   (4) 57° C. for 30 seconds;    -   (5) 95° C. for 30 seconds;    -   (6) 64° C. for 30 seconds;    -   (7) 95° C. for 30 seconds;    -   (8) Repeat steps 6 and 7 thirty nine (39) times;    -   (9) 72° C. for 5 minutes.

In the first cycle of PCR, the annealing temperature was about themelting temperature of the 3′ annealing region of the second primers,which was 37° C. The annealing temperature in the second cycle of PCRwas about the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the first primer, which was 57° C. The annealingtemperature in the third cycle of PCR was about the melting temperatureof the entire sequence of the second primer, which was 64° C. Theannealing temperature for the remaining cycles was 64° C. Escalating theannealing temperature from TM1 to TM2 to TM3 in the first three cyclesof PCR greatly improves specificity. These annealing temperatures arerepresentative, and the skilled artisan will understand the annealingtemperatures for each cycle are dependent on the specific primers used.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results.

Purification of Fragment of Interest

The PCR products were separated from the unused PCR reagents. After thePCR reaction, ½ of the reaction volume for SNP TSC0903430, SNPTSC0337961, and SNP TSC0786441 were mixed together in a single reactiontube. One-half the reaction volumes for SNPs TSC1168303, TSC0056188,TSC0466177, and TSC0197424 were pooled together in a single reactiontube. The un-used primers, and nucleotides were removed from thereaction by using Qiagen MinElute PCR purification kits (Qiagen, CatalogNumber 28004). The reactions were performed following the manufacturer'sinstructions supplied with the columns.

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme BsmFI, which binds to the recognition site incorporated into the PCRproducts from the second primer. The digests were performed in eppendorftubes following the instructions supplied with the restriction enzyme.

Incorporation of Labeled Nucleotide

The restriction enzyme digest with BsmF I yielded a DNA fragment with a5′ overhang, which contained the SNP site or locus of interest and a 3′recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide or nucleotides in the presence of a DNApolymerase.

As discussed in detail in Example 6, the sequence of both alleles of aSNP can be determined with one labeled nucleotide in the presence of theother unlabeled nucleotides. The following components were added to eachfill in reaction: 1 μl of fluorescently labeled ddGTP, 0.5 μl ofunlabeled ddNTPs (40 μM), which contained all nucleotides exceptguanine, 2 μl of 10× sequenase buffer, 0.25 μl of Sequenase, and wateras needed for a 20 μl reaction. The fill in reaction was performed at40° C. for 10 min. Sequenase was the DNA polymerase used in thisexample. However, any DNA polymerase can be used for a fill-in reactionincluding but not limited to E. coli DNA polymerase, Klenow fragment ofE. coli DNA polymerase I, T7 DNA polymerase, T4 DNA polymerase, Tagpolymerase, Pfu DNA polymerase, Vent DNA polymerase, polymerase frombacteriophage 29, and REDTaq™ Genomic DNA polymerase. Non-fluorescentlylabeled ddNTP was purchased from Fermentas Inc. (Hanover, Md.). An otherlabeling reagents were obtained from Amersham (Thermo Sequenase DyeTerminator Cycle Sequencing Core Kit, US 79565).

Detection of the Locus of Interest

The sample was loaded into a lane of a 36 cm 5% acrylamide (urea) gel(BioWhittaker Molecular Applications, Long Ranger Run Gel Packs, catalognumber 50691). The sample was electrophoresed into the gel at 3000 voltsfor 3 min. The gel was run for 3 hours on a sequencing apparatus (HoeferSQ3 Sequencer). The gel was removed from the apparatus and scanned onthe Typhoon 9400 Variable Mode Imager. The incorporated labelednucleotide was detected by fluorescence.

Below, a schematic of the 5′ overhang for SNP TSC0056188 is reproduced(where R indicates the variable site). The entire sequence is not shown,only a portion of the overhang.

5′CCA 3′GGT R T C C Overhang position 1 2 3 4

As discussed in detail in Example 6, one nucleotide labeled with onechemical moiety can be used to determine the sequence of the alleles ofa locus of interest. The observed nucleotides for TSC0056188 on the 5′sense strand (here depicted as the top strand) are adenine and guanine.The third position in the overhang on the antisense strand is cytosine,which is complementary to guanine. As the variable site can be adenineor guanine, fluorescently labeled ddGTP in the presence of unlabeleddCTP, dTTP, and dATP was used to determine the sequence of both alleles.The fill-in reactions for an individual homozygous for guanine,homozygous for adenine or heterozygous are diagrammed below.

Homozygous Adenine:

5′CCA A A G* 3′GGT T T C C Overhang position 1 2 3 4

Homozygous Guanine:

5′CCA G* 3′GGT C T C C Overhang position 1 2 3 4

Heterozygous:

Allele 1 5′CCA G* 3′GGT C T C C Overhang position 1 2 3 4 Allele 2 5′CCAA A G* 3′GGT T T C C Overhang position 1 2 3 4

As seen in FIG. 14, two bands were detected for SNP TSC0056188. Thelower band corresponded to DNA molecules tilled in with ddGTP atposition one complementary to the overhang, which is representative ofthe guanine allele. The higher band, separated by a single base from thelower band, corresponded to DNA molecules filled in with ddGTP atposition 3 complementary to the overhang. This band represented theadenine allele. The intensity of each band was strong, indicating thateach allele was well represented in the population. SNP TSC0056188 isrepresentative of a SNP with high allele frequency.

Below, a schematic of the 5′ overhang generated after digestion withBsmF I for SNP TSC0337961 is reproduced (where R indicates the variablesite). The entire sequence is not shown, only a portion of the overhang.

5′ GCCA 3′ CGGT R G C T Overhang position 1 2 3 4

The observed nucleotides for SNP TSC0337961 on the 5′ sense strand (heredepicted as the top strand) are adenine and guanine. The third positionin the overhang on the antisense strand was cytosine, which iscomplementary to guanine. As the variable site can be adenine orguanine, fluorescently labeled ddGTP in the presence of unlabeled dCTP,dTTP, and dATP was used to determine the sequence of both alleles. Thefill-in reactions for an individual homozygous for guanine, homozygousfor adenine or heterozygous are diagrammed below.

Homozygous for Guanine:

5′ GCCA G* 3′ CGGT C G C T Overhang position 1 2 3 4

Homozygous for Adenine:

5′ GCCA A C G* 3′ CGGT T G C T Overhang position 1 2 3 4

Heterozygous

Allele 1 5′ GCCA G* 3′ CGGT C G C T Overhang position 1 2 3 4 Allele 25′ GCCA A C G* 3′ CGGT T G C T Overhang position 1 2 3 4

As seen in FIG. 14, one band migrating at the position of the expectedlower molecular weight band was observed. This band represented the DNAmolecules filled in with ddGTP at position one complementary to theoverhang, which represents the guanine allele. No band corresponding tothe DNA molecules filled in with ddGTP at position 3 complementary tothe overhang was detected. SNP TSC0337961 is representative of a SNPthat is not highly variable within the population.

Of the seven SNPs analyzed, four of the SNPs (TSC1168303, TSC0056188,TSC0466177, and TSC0197424 had high allele frequencies. Two bands ofhigh intensity were seen for each of the four SNPs, indicating that bothalleles were well represented in the population.

However, it is not necessary that the SNPs have allele frequencies of50:50 to be useful. All SNPs provide useful information. The methodsdescribed herein provide a rapid technique for determining the allelefrequency of a SNP, or any variable site including but not limited topoint mutations. Allele frequencies of 50:50, 51:49, 52:48, 53:47,54:46, 55:45, 56:46, 57:43, 58:42, 59:41, 60:40, 61:39, 62:38, 63:37,64:36, 65:35, 66:34, 67:33, 68:32, 69:31, 70:30, 71:29, 72:28, 73:27,74:26, 75:25, 76:24, 77:23, 78:22, 79:21, 80:20, 81:19, 82:18, 83:17,84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6,95:5, 96:4, 97:3, 98:2, 99:1 and 100:0 can be useful.

Two bands were seen for SNP TSC0903430. One band, the lower molecularweight band represented the DNA molecules filled in with labeled ddGTP.A band of weaker intensity was seen for the molecules filled in withlabeled ddGTP at position 3 complementary to the overhang, whichrepresented the cytosine allele. SNP TSC0903430 represents a SNP withlow allele frequency variation. In the population, the majority ofindividuals carry the guanine allele, but the cytosine allele is stillpresent.

One band of high intensity was seen for SNP TSC0337961 and SNPTSC0786441. The band detected for both SNP TSC0337961 and SNP TSC0786441corresponded to the DNA molecules filled in with ddGTP at position 1complementary to the overhang. No signal was detected from DNA moleculesthat would have been filled in at position 3 complementary to theoverhang, which would have represented the second allele. SNP TSC0337961and SNP TSC0786441 represent SNPs with little variability in thepopulation.

As demonstrated in FIG. 14, the first primer used to amplify each locusof interest can be designed to anneal at various distances from thelocus of interest. This allows multiple SNPs to be analyzed in the samereaction. By designing the first primer to anneal at specified distancesfrom the loci of interest, any number of loci of interest can beanalyzed in a single reaction including but not limited to 1-10, 11-20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110,111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190,191-200, 201-300, 301-400, 401-500, and greater than 500.

As discussed in Example 6, some type IIs restriction enzymes displayalternate cutting patterns. For example, the type IIS restriction enzymeBsmF I typically cuts 10/14 from its binding site; however, the enzymealso can cut 11/15 from the binding site. To eliminate the effect of thealternate cut, the labeled nucleotide used for the fill-in reactionshould be chosen such that it is not complementary to position 0 of theoverhang generated by the 11/15 cut (discussed in detail in Example 6).For instance, if you label with ddGTP, the nucleotide preceding thevariable site on the strand that is filled in should not be a guanine.

The 11/15 overhang generated by BsmF I or SNP TSC0056188 is depictedbelow, with the variable site in bold-typeface:

Allele 1 5′CC 3′GG T C T C Overhang position 0 1 2 3 Allele 2 5′CC 3′GGT T T C Overhang position 0 1 2 3

After the fill-in reaction with labeled ddGTP, unlabeled dATP, dTTP, anddCTP, the following molecules were generated:

11/15 Allele 1 5′CC A G* 3′GG T C T C Overhang position 0 1 2 3 11/15Allele 2 5′CC A A A G* 3′GG T T T C Overhang position 0 1 2 3

Two signals were seen; one band corresponded to molecules filled in withddGTP at position one of the overhang, and the other band correspondedto the molecules filled in with ddGTP at position 3 complementary to theoverhang. These are the same DNA molecules generated after the fill-inreaction of the 10/14 overhang. Thus, the two bands can be comparedwithout any ambiguity from the alternate cut. This method of labelingwith a single nucleotide eliminates any errors generated from thealternate cutting properties of the enzymes.

The methods described herein is applicable to determining the allelefrequency of any SNP including but not limited to SNPs on humanchromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, X and Y.

Example 9

Heterozygous SNPs, by definition, differ by one nucleotide. At aheterozygous SNP, allele 1 and allele 2 may be present at a ratio of1:1. However, it is possible that DNA polymerases can incorporate onenucleotide at a faster rate than other nucleotides, and thus theobserved ratio of a heterozygous SNP may differ from the theoreticallyexpected 1:1 ratio.

Below, methods are described that allow efficient and accuratequantitation for the expected ratio of allele 1 to allele 2 at aheterozygous SNP.

Preparation of Template DNA

Template DNA was obtained from twenty-four individuals after informedconsent had been granted. From each individual, a 9 ml blood sample wascollected into a sterile tube (Fischer Scientific, 9 ml EDTA Vacuettetubes, catalog number NC9897284). The tubes were spun at 1000 rpm forten minutes without brake. The supernatant (the plasma) of each samplewas removed, and one milliliter of the remaining blood sample, which iscommonly referred to as the “buffy-coat” was transferred to a new tube.One milliliter of 1×PBS was added to each sample.

Template DNA was isolated using the Q1 Amp DNA Blood Midi Kit suppliedby QIAGEN (Catalog number 51183). The template DNA was isolated as perinstructions included in the kit.

Design of Primers

SNP TSC0607185 was amplified using the following primer set:

First primer: (SEQ ID NO: 40) 5′ ACTTGATTCCGTGAATTCGTTATCAATAAATCTTACAT3′ Second primer: (SEQ ID NO: 41) 5′ CAAGTTGGATCCGGGACCCAGGGCTAACC 3′

SNP TSC1130902 was amplified using the following primer set:

First primer: (SEQ ID NO: 34) 5′ TCTAACCATTGCGAATTCAGGGCAAGGGGGGTGAGATC3′ Second primer: (SEQ ID NO: 35) 5′ TGACTTGGATCCGGGACAACGACTCATCC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI. The second primer contained therecognition site for the restriction enzyme BsmF I. The first primer wasdesigned to anneal at various distances from the locus of interest.

The first primer for SNP TSC0607185 was designed to anneal ninety basesfrom the locus of interest. The first primer for SNP TSC1130902 wasdesigned to anneal sixty bases from the locus of interest.

All loci of interest were amplified from the template genomic DNA usingthe polymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and4,683,202, incorporated herein by reference). In this example, the lociof interest were amplified in separate reaction tubes but they couldalso be amplified together in a single PCR reaction. For increasedspecificity, a “hot-start” PCR was used. PCR reactions were performedusing the HotStarTaq Master Mix Kit supplied by QIAGEN (catalog number203443). The amount of template DNA and primer per reaction can beoptimized for each locus of interest but in this example, 40 ng oftemplate human genomic DNA and 5 μM of each primer were used. Fortycycles of PCR were performed. The following PCR conditions were used:

-   -   (1) 95° C. for 15 minutes and 15 seconds;    -   (2) 37° C. for 30 seconds;    -   (3) 95° C. for 30 seconds;    -   (4) 57° C. for 30 seconds;    -   (5) 95° C. for 30 seconds;    -   (6) 64° C. for 30 seconds;    -   (7) 95° C. for 30 seconds;    -   (8) Repeat steps 6 and 7 thirty nine (39) times;    -   (9) 72° C. for 5 minutes.

In the first cycle of PCR, the annealing temperature was about themelting temperature of the 3′ annealing region of the second primers,which was 37° C. The annealing temperature in the second cycle of PCRwas about the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the first primer, which was 57° C. The annealingtemperature in the third cycle of PCR was about the melting temperatureof the entire sequence of the second primer, which was 64° C. Theannealing temperature for the remaining cycles was 64° C. Escalating theannealing temperature from TM1 to TM2 to TM3 in the first three cyclesof PCR greatly improves specificity. These annealing temperatures arerepresentative, and the skilled artisan will understand the annealingtemperatures for each cycle are dependent on the specific primers used.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results.

Purification of Fragment of Interest

The PCR products were separated from the genomic template DNA. One halfof the PCR reaction was transferred to a well of a Streptawell,transparent, High-Bind plate from Roche Diagnostics GmbH (catalog number1 645 692, as listed in Roche Molecular Biochemicals, 2001 BiochemicalsCatalog). The first primers contained a 5′ biotin tag so the PCRproducts bound to the Streptavidin coated wells while the genomictemplate DNA did not. The streptavidin binding reaction was performedusing a Thermomixer (Eppendorf) at 1000 rpm for 20 min. at 37° C. Eachwell was aspirated to remove unbound material, and washed three timeswith 1×PBS, with gentle mixing (Kandpal et al., Nucl. Acids Res.18:1789-1795 (1990); Kaneoka et al., Biotechniques 10:30-34 (1991);Green et al., Nucl. Acids Res. 18:6163-6164 (1990)).

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme BsmFI, which binds to the recognition site incorporated into the PCRproducts from the second primer. The digests were performed in theStreptawells following the instructions supplied with the restrictionenzyme. After digestion, the wells were washed three times with PBS toremove the cleaved fragments.

Incorporation of Labeled Nucleotide

The restriction enzyme digest with BsmF I yielded a DNA fragment with a5′ overhang, which contained the SNP site or locus of interest and a 3′recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide or nucleotides in the presence of a DNApolymerase.

As discussed in detail in Example 6, the sequence of both alleles of aSNP can be determined by using one labeled nucleotide in the presence ofthe other unlabeled nucleotides. The following components were added toeach fill in reaction: 1 μl of fluorescently labeled ddGTP, 0.5 μl ofunlabeled ddNTPs (40 μM), which contained all nucleotides exceptguanine, 2 μl of 10× sequenase buffer, 0.25 μl of Sequenase, and wateras needed for a 20 μl reaction. The fill in reaction was performed at40° C. for 10 min. Non-fluorescently labeled ddNTP was purchased fromFermentas Inc. (Hanover, Md.). All other labeling reagents were obtainedfrom Amersham (Thermo Sequenase Dye Terminator Cycle Sequencing CoreKit, US 79565).

After labeling, each Streptawell was rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments were then released from theStreptawells by digestion with the restriction enzyme EcoRI, accordingto the manufacturer's instructions that were supplied with the enzyme.Digestion was performed for 1 hour at 37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

The samples were loaded into a lane of a 36 cm 5% acrylamide (urea) gel(BioWhittaker Molecular Applications, Long Ranger Run Gel Packs, catalognumber 50691). The samples were electrophoresed into the gel at 3000volts for 3 min. The gel was run for 3 hours on a sequencing apparatus(Hoefer SQ3 Sequencer). The gel was removed from the apparatus andscanned on the Typhoon 9400 Variable Mode Imager. The incorporatedlabeled nucleotide was detected by fluorescence. A box was drawn aroundeach band and the intensity of the band was calculated using the Typhoon9400 Variable Mode Imager software.

Below, a schematic of the 5′ overhang for SNP TSC0607185 is shown. Theentire DNA sequence is not reproduced, only the portion to demonstratethe overhang (where R indicates the variable site).

C C T R TGTC 3′ ACAG 5′ 4 3 2 1 Overhang position

The observed nucleotides at the variable site for TSC0607185 on the 5′sense strand (here depicted as the top strand) are cytosine andthymidine (depicted here as R). In this case, the second primer annealsfrom the locus of interest, which allows the fill-in reaction to occuron the anti-sense strand (depicted here as the bottom strand). Theantisense strand will be filled in with guanine or adenine.

The second position in the 5′ overhang is thymidine, which iscomplementary to adenine, and the third position in the overhangcorresponds to cytosine, which is complementary to guanine.Fluorescently labeled ddGTP in the presence of unlabeled dCTP, dTTP, anddATP was used to determine the sequence of both alleles. After thefill-in reaction, the following DNA molecules were generated:

C C T C TGTC 3′ Allele 1 G* ACAG 5′ 4 3 2 1 Overhang position C C T TTGTC 3′ Allele 1 G* A A ACAG 5′ 4 3 2 1 Overhang position

The overhang generated by BsmF I cutting at 11/15 from the recognitionsite at TSC0607185 is depicted below:

C T R T GTC 3′ 11/15 CAG 5′ 3 2 1 0 Overhang position

As labeled ddGTP is used for the fill-in reaction, no new signal will begenerated from the molecules cut 11/15 from the recognition site.Position 0 complementary to the overhang was filled in with unlabeleddATP. Only signals generated from molecules filled in with labeled ddGTPat position 1 complementary to the overhang or molecules filled in withlabeled ddGTP at position 3 complementary to the overhang were seen.

Five of the twenty-four individuals were heterozygous for SNPTSC0607185. As shown in FIG. 15, two bands were detected. The lowermolecular weight band corresponded to DNA molecules filled in with ddGTPat position 1 complementary to the overhang. The higher molecular weightband corresponded to DNA molecules filled in with ddGTP at position 3complementary to the overhang.

The ratio of the two alleles was calculated for each of the fiveheterozygous samples (see Table XVI). The average ratio of allele 2 toallele 1 was 1.000 with a standard deviation of 0.044. Thus, the alleleratio at SNP TSC0607185 was highly consistent. The experimentallycalculated allele ratio for a particular SNP is hereinafter referred toas the “p” value of the SNP. Analysis of SNP TSC0607185 consistentlywill provide an allele ratio of 1:1, provided that the number of genomesanalyzed is of sufficient quantity that no error is generated fromstatistical sampling.

If the sample contained a low number of genomes, it is statisticallypossible that the primers will anneal to one chromosome over anotherchromosome. For example, if the sample contains 40 genomes, whichcorresponds to a total of 40 chromosomes of allele 1 and 40 chromosomesof allele 2, the primers may anneal to 40 chromosomes of allele 1 butonly 35 chromosome of allele 2. This would cause allele 1 to beamplified preferentially to allele 2, which would alter the ratio ofallele 1 to allele 2. This problem is eliminated by having a sufficientnumber of genomes in the sample.

SNP TSC0607185 represents a SNP where the difference in the nucleotideat the variable site does not affect the PCR reaction, or digestion withthe restriction enzyme or the fill-in reaction. The use of onenucleotide labeled with one fluorescent dye assures that the bands forone allele can be accurately compared to the bands for the secondallele. There is no added complication of having to compare between twodifferent lanes, or having to correct for the quantum coefficients ofthe dyes. Additionally, any effect from the alternate cutting propertiesof the type IIS restriction enzymes has been removed.

TABLE XVI Ratio of allele 2 to allele 1 at SNPs TSC0607185 andTSC1130902. SNP TSC0607185 SNP TSC1130902 Sample Allele 1 Allele 2Allele2/Allele1 Allele 1 Allele 2 Allele2/Allele 1 1 2382 2313 0.9710335877 4433 0.754296 2 1581 1533 0.969639 3652 2695 0.737952 3 1795 18791.046797 5416 3964 0.730059 4 1921 1855 0.965643 3493 2663 0.762382 51618 1701 1.051298 3894 2808 0.721109 Average 1.000882 0.74116 STD0.044042 0.017018

Below, a schematic of the 5′ overhang for SNP TSC1130902 is shown. Theentire DNA sequence is not reproduced, only the portion to demonstratethe overhang (where R indicates the variable site).

5′ TTCAT 3′ AAGTA R T C C Overhang position 1 2 3 4

The observed nucleotides for TSC1130902 on the 5′ sense strand (heredepicted as the top strand) are adenine and guanine. The second positionin the overhang corresponds to a thymidine, and the third position inthe overhang corresponds to cytosine, which is complementary to guanine.Fluorescently labeled ddGTP in the presence of unlabeled dCTP, dTTP, anddATP was used to determine the sequence of both alleles. After thefill-in reaction, the following DNA molecules were generated:

Allele 1 5′ TTCAT G* 3′ AAGTA C T C C Overhang position 1 2 3 4 Allele 25′ TTCAT A A G* 3′ AAGTA T T C C Overhang position 1 2 3 4

As shown in FIG. 15, two bands were detected. The lower molecular weightband corresponded to DNA molecules filled in with labeled ddGTP atposition 1 complementary to the overhang (the G allele). The highermolecular weight band, separated by a single base from the lower band,corresponded to DNA molecules filled in with ddGTP at position 3complementary to the overhang (the A allele).

Five of the twenty-four individuals were heterozygous for SNPTSC1130902. As seen in FIG. 15, the band corresponding to allele 1 wasmore intense than the band corresponding to allele 2. This was seen foreach of the five individuals. The actual intensity of the bandcorresponding to allele 1 varied from individual to individual but itwas always more intense than the band corresponding to allele 2. For thefive individuals, the average ratio of allele 2 to allele 1 was 0.74116,with a standard deviation of 0.017018.

Template DNA was prepared from five different individuals. Separate PCRreactions, separate restriction enzyme digestions, and separate fill-inreactions were performed. However, for each template DNA, the ratio ofallele 2 to allele 1 was about 0.75. The “p” value for this SNP washighly consistent.

For example, for SNP TSC1130902, the “p” value was 0.75. Any deviationfrom this value, provided the sample contains an adequate number ofgenomes to remove statistical sampling errors, will indicate that thereis an abnormal copy number of chromosome 13. If there is an additionalcopy of allele 2, the “p” value will be higher than the expected 0.75.However, if there is an addition copy of allele 1, the “p” value will belower than the expected 0.75. With the “p” value quantitated for aparticular SNP, that SNP can be used to determine the presence orabsence of a chromosomal abnormality. An accurate “p” value measured fora single SNP will be sufficient to detect the presence of a chromosomalabnormality.

There are several possible explanations for why the ratio of one alleleto the other allele at some SNPs varies from the theoretically expectedratio of 1:1. First, it is possible that the DNA polymerase incorporatesone nucleotide faster than the other nucleotide. As the alleles arebeing amplified by PCR, even a slight preference for one nucleotide overthe other may cause variation from the expected 1:1 ratio. Thispotential preference for one nucleotide over the other is not seenduring the fill-in reaction because a single nucleotide labeled with onedye is used.

It is also possible that the variable nucleotide at the SNP siteinfluences the rate of denaturation of the two alleles. If allele 1contains a guanine and allele 2 contains an adenine, the differencebetween the strength of the bonds for these nucleotides may affect therate at which the DNA strands separate. Again, it is important tomention that the alleles are being amplified by PCR so very subtledifferences can make a large impact on the final result. It is alsopossible that the variable nucleotide at the SNP site influences therate at which the two strands anneal after separation.

Alternatively, it is possible that the type IIS restriction enzyme cutsone allele preferentially to the other allele. As discussed in detailabove, type IIS restriction enzymes cut at a distance from therecognition site. It is possible that the variable nucleotide at the SNPsite influences the efficiency of the restriction enzyme digestion. Itis possible that at some SNPs the restriction enzyme cuts one allelewith an efficiency of 100%, while it cuts the other allele with anefficiency of 90%.

However, the fact that the ratio of allele 1 to allele 2 deviates fromthe theoretically expected ratio of 1:1, does not influence or reducethe utility of that SNP. As demonstrated above, the “p” value for eachSNP is consistent among different individuals.

The “p” value for any SNP can be calculated by analyzing the templateDNA of any number of heterozygous individuals including but not limitedto 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100,101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180,181-190, 191-200, 201-210, 211-220, 221-230, 231-240, 241-250, 251-260,261-270, 271-280, 281-290, 291-300, and greater than 300.

The methods described herein allow the “p” value for any SNP to bedetermined. It is possible that some SNPs will behave more consistentlythan other SNPs. In the human genome, there are over 3 million SNPs; itis not possible to speculate on how each SNP will behave. The “p” valuefor each SNP will have to be experimentally determined. The methodsdescribed herein allow identification of SNPs that have highlyconsistent, and reproducible “p” values.

Example 10

As discussed in Example 9, the ratio of one allele to the other alleleat a particular SNP may vary from the theoretically expected ratio of50:50. These SNPs can be used to detect the presence of additionalchromosomes provided that the ratio of one allele to the other alleleremains linear in individuals with chromosomal disorders. For example,at SNP X if the percentage of allele 1 to allele 2 is 75:25, theexpected percentage of allele 1 to allele 2 for an individual withDown's syndrome must be properly adjusted to reflect the variation fromthe expected percentage at this SNP.

The percentage of allele 1 to allele 2 for SNP TSC0108992 on chromosome21 was calculated using template DNA from four normal individuals andtemplate DNA from an individual with Down's syndrome. As demonstratedbelow, the percentage of one allele to the other allele was consistentand remained linear in an individual with Down's syndrome.

Preparation of Template DNA

DNA was obtained from four individuals with a normal genetic karyotypeand an individual identified as having an extra copy of chromosome 21(Down's syndrome). Informed consent was obtained from all individuals.Informed consent also was obtained from the parents of the individualwith Down's syndrome.

From each individual, a 9 ml blood sample was collected into a steriletube (Fischer Scientific, 9 ml EDTA Vacuette tubes, catalog numberNC9897284). Template DNA was isolated using the QIAmp DNA Blood Midi Kitsupplied by QIAGEN (Catalog number 51183). The template DNA was isolatedas per instructions included in the kit.

Design of Primers

SNP TSC0108992 was amplified using the following primer set:

First primer: (SEQ ID NO: 293) 5′ CTACTGAGGGCTCGTAGATCCCAATTCCTTCCCAAGCT3′ Second primer: (SEQ ID NO: 294) 5′ AATCCTGCTTTAGGGACCATGCTGGTGGA 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC0108992 was amplified from the template genomic DNA using thepolymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202,incorporated herein by reference). For increased specificity, a“hot-start” PCR was used. PCR reactions were performed using theHotStarTaq Master Mix Kit supplied by QIAGEN (catalog number 203443).The amount of template DNA and primer per reaction can be optimized foreach locus of interest. In this example, 50 ng of template human genomicDNA and 5 μM of each primer were used. Thirty-eight cycles of PCR wereperformed. The following PCR conditions were used:

-   -   (1) 95° C. for 15 minutes and 15 seconds;    -   (2) 37° C. for 30 seconds;    -   (3) 95° C. for 30 seconds;    -   (4) 57° C. for 30 seconds;    -   (5) 95° C. for 30 seconds;    -   (6) 64° C. for 30 seconds;    -   (7) 95° C. for 30 seconds;    -   (8) Repeat steps 6 and 7 thirty-seven (37) times;    -   (9) 72° C. for 5 minutes.

In the first cycle of PCR, the annealing temperature was about themelting temperature of the 3′ annealing region of the second primers,which was 37° C. The annealing temperature in the second cycle of PCRwas about the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the first primer, which was 57° C. The annealingtemperature in the third cycle of PCR was about the melting temperatureof the entire sequence of the second primer, which was 64° C. Theannealing temperature for the remaining cycles was 64° C. Escalating theannealing temperature from TM1 to TM2 to TM3 in the first three cyclesof PCR greatly improves specificity. These annealing temperatures arerepresentative, and the skilled artisan will understand the annealingtemperatures for each cycle are dependent on the specific primers used.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results.

Purification of Fragment of Interest

The PCR products were separated from the genomic template DNA. Each PCRreaction was split into two samples and transferred to two separatewells of a Streptawell, transparent, High-Bind plate from RocheDiagnostics GmbH (catalog number 1 645 692, as listed in Roche MolecularBiochemicals, 2001 Biochemicals Catalog). For each PCR reaction, therewere two replicates; each in a separate well of a microtiter plate. Thefirst primer contained a 5′ biotin tag so the PCR products bound to theStreptavidin coated wells while the genomic template DNA did not. Thestreptavidin binding reaction was performed using a Thermomixer(Eppendorf) at 1000 rpm for 20 min. at 37° C. Each well was aspirated toremove unbound material, and washed three times with 1×PBS, with gentlemixing (Kandpal et al., Nucl. Acids Res. 18:1789-1795 (1990); Kaneoka etal., Biotechniques 10:30-34 (1991); Green et al., Nucl. Acids Res.18:6163-6164 (1990)).

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme BsmFI, which binds to the recognition site incorporated into the PCRproducts from the second primer. The digests were performed in theStreptawells following the instructions supplied with the restrictionenzyme. After digestion, the wells were washed three times with 1×PBS toremove the cleaved fragments.

Incorporation, of Labeled Nucleotide

The restriction enzyme digest with BsmF I yielded a DNA fragment with a5′ overhang, which contained the SNP site or locus of interest and a 3′recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide or nucleotides in the presence of a DNApolymerase.

As discussed in detail in Example 6, the sequence of both alleles of aSNP can be determined with one labeled nucleotide in the presence of theother unlabeled nucleotides. The following components were added to eachfill in reaction: 1 μl of fluorescently labeled ddTTP, 0.5 μl ofunlabeled ddNTPs (40 μM), which contained all nucleotides exceptthymidine, 2 μl of 10× sequenase buffer, 0.25 μl of Sequenase, and wateras needed for a 20 μl reaction. The fill in reaction was performed at40° C. for 10 min. Non-fluorescently labeled ddNTP was purchased fromFermentas Inc. (Hanover, Md.). All other labeling reagents were obtainedfrom Amersham (Thermo Sequenase Dye Terminator Cycle Sequencing CoreKit, US 79565).

After labeling, each Streptawell was rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments were then released from theStreptawells by digestion with the restriction enzyme EcoRI, accordingto the manufacturer's instructions that were supplied with the enzyme.Digestion was performed for 1 hour at 37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

The samples were loaded into the lanes of a 36 cm 5% acrylamide (urea)gel (BioWhittaker Molecular Applications, Long Ranger Run Gel Packs,catalog number 50691). The samples were electrophoresed into the gel at3000 volts for 3 min. The gel was run for 3 hours on a sequencingapparatus (Hoefer SQ3 Sequencer). The gel was removed from the apparatusand scanned on the Typhoon 9400 Variable Mode Imager. The incorporatedlabeled nucleotide was detected by fluorescence. A box was drawn aroundeach band and the intensity of the band was calculated using the Typhoon9400 Variable Mode Imager software.

Below, a schematic of the 5′ overhang for SNP TSC0108992 is shown. Theentire DNA sequence is not reproduced, only the portion to demonstratethe overhang (where R indicates the variable site).

GTCC 3′ G A C R CAGG 5′ 4 3 2 1 Overhang Position

The observed nucleotides for SNP TSC0108992 are adenine and thymidine onthe sense strand (here depicted as the top strand). Position 3 of theoverhang corresponds to adenine, which is complementary to thymidine.Labeled ddTTP was used in the presence of unlabeled dATP, dCTP, anddGTP. After the fill-in reaction with labeled ddTTP, the following DNAmolecules were generated:

T* G A GTCC 3′ Allele 1 G A C T CAGG 5′ 4 3 2 1 Overhang Position T*GTCC 3′ Allele 2 G A C A CAGG 5′ 4 3 2 1 Overhang Position

There was no difficulty in comparing the values obtained from allele 1to allele 2 because one labeled nucleotide was used for the fill-inreaction, and the fill-in reaction for both alleles occurred in a singletube. The alternate cutting properties of BsmF I would not influencethis analysis because the 11/15 overhang would be filled in just as the10/14 overhang. Schematics of the filled-in 11/15 overhangs are depictedbelow:

T* G A G TCC 3′ 11/15 Allele 1 A C T C AGG 5′ 3 2 1 0 Overhang PositionT* G TCC 3′ 11/15 Allele 2 A C A C AGG 5′ 3 2 1 0 Overhang Position

As seen in FIG. 16, two bands were seen for each sample of template DNA.The lower molecular weight band corresponded to the DNA molecules filledin with ddTTP at position one complementary to the overhang, and thehigher molecular weight band corresponded to DNA molecules filled inwith ddTTP at position 3 complementary to the overhang.

The percentage of allele 2 to allele 1 was highly consistent. (see TableXVII). In addition, for any given individual, the replicates of the PCRreaction showed similar results (see Table XVII). The percentage ofallele 2 to allele 1 was calculated by dividing the value of allele 2 bythe sum of the values for allele 1 and allele 2 (allele 2/(allele1+allele 2)). From four individuals, the average percentage of allele 2to allele 1 was 0.4773 with a standard deviation of 0.0097. Thepercentage of allele 2 to allele 1 on template DNA isolated from anindividual with Down's syndrome was 0.3086.

The theoretically expected percentage of allele 2 to allele 1 usingtemplate DNA from a normal individual is 0.50. However, theexperimentally determined percentage was 0.4773. The theoreticallyexpected percentage of allele 2 to allele 1 for an individual with anextra copy of chromosome 21 is 0.33. The experimentally determinedpercentage of allele 2 to allele 1 for SNP TSC0108992 was 0.3086.

The deviation from the theoretically expected percentage is highlyconsistent and remains linear. The following formula demonstrates thatthe percentage of allele 2 to allele 1 at SNP TSC0108992 remains lineareven on template DNA obtained from an individual with an extra copy ofchromosome 21:

$\frac{0.47}{0.50} = \frac{X}{0.33}$ X = 0.3102

If the percentage of allele 2 to allele 1 using template DNA obtainedfrom a normal individual is determined to be 0.47, then the percentageof allele 2 to allele 1 using template DNA from an individual withDown's syndrome should be 0.3102. The experimentally determined ratiowas 0.3086, with a standard deviation of 0.00186. There is no differencebetween the predicted percentage and the experimentally determinedpercentage of allele 2 to allele 1 on template DNA from an individualwith Down's syndrome.

The percentage of one allele to the other allele at a particular SNP ishighly consistent, reproducible, and linear. This demonstrates that anySNP, regardless of the calculated percentage for one allele to another,can be used to determine the presence or absence of a chromosomaldisorder.

TABLE XVII Percentage of Allele 2 to Allele 1 at SNP TSC0108992. SampleAllele 2 Allele 1 2/(2 + 1) 1A 9568886 10578972 0.474933 1B 83308649221381 0.474632 2A 9801053 10345444 0.486489 2B 8970942 96031020.482983 3A 8676718 9211085 0.485063 3B 10847024 11420943 0.487113 4A10512420 12227107 0.462297 4B 7883584 9055289 0.465414 MEAN 0.477366STDEV 0.009654 DS 6797400 15138959 0.309869 DS 6025753 13586890 0.307238MEAN 0.308554 STDEV 0.00186

Example 11

The percentage of allele 2 to allele 1 for a particular SNP is highlyconsistent. Statistically significant deviation from the experimentallydetermined ratio indicates the presence of a chromosomal abnormality.Below, the percentage of allele 2 to allele 1 at SNP TSC0108992 onchromosome 21 was calculated using template DNA from a normal individualand template DNA from an individual with Down's syndrome. Mixturescontaining various amounts of normal DNA and Down's syndrome DNA wereprepared and analyzed in a blind fashion.

Preparation of Template DNA

DNA was obtained from an individual with a normal genetic karyotype andan individual identified as having an extra copy of chromosome 21(Down's syndrome). Informed consent was obtained from both individuals.Informed consent also was obtained from the parents of the individualwith Down's syndrome.

From each individual, a 9 ml blood sample was collected into a steriletube (Fischer Scientific, 9 ml EDTA Vacuette tubes, catalog numberNC9897284). Template DNA was isolated using the QIAmp DNA Blood Midi Kitsupplied by QIAGEN (Catalog number 51183). The template DNA was isolatedas per instructions included in the kit.

Mixtures of Template DNA

The template DNA from the individual with the normal karyotype and thetemplate DNA from the individual with an extra copy of chromosome 21were diluted to a concentration of 10 ng/μl. Four mixtures of normaltemplate DNA and Down's syndrome template DNA were made in the followingfashion:

Mixture 1: 32 μl of Normal DNA+8 μl of Down's syndrome DNA

Mixture 2: 28 μl of Normal DNA+12 μl of Down's syndrome DNA

Mixture 3: 20 μl of Normal DNA+20 μl of Down's syndrome DNA

Mixture 4: 10 μl of Normal DNA+30 μl of Down's syndrome DNA

Three separate PCR reactions were set up for the normal template DNA andthe template DNA from the individual with Down's syndrome. Likewise, foreach mixture, three separate PCR reactions were set up.

Design of Primers

SNP TSC0108992 was amplified using the following primer set:

First primer: (SEQ ID NO: 293) 5′ CTACTGAGGGCTCGTAGATCCCAATTCCTTCCCAAGCT3′ Second primer: (SEQ ID NO: 294) 5′ AATCCTGCTTTAGGGACCATGCTGGTGGA 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC0108992 was amplified from the template genomic DNA using thepolymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202,incorporated herein by reference).

For increased specificity, a “hot-start” PCR was used. PCR reactionswere performed using the HotStarTaq Master Mix Kit supplied by QIAGEN(catalog number 203443). The amount of template DNA and primer perreaction can be optimized for each locus of interest but in thisexample, 50 ng of template human genomic DNA and 5 μM of each primerwere used. Thirty-eight cycles of PCR were performed. The following PCRconditions were used:

-   -   (1) 95° C. for 15 minutes and 15 seconds;    -   (2) 37° C. for 30 seconds;    -   (3) 95° C. for 30 seconds;    -   (4) 57° C. for 30 seconds;    -   (5) 95° C. for 30 seconds;    -   (6) 64° C. for 30 seconds;    -   (7) 95° C. for 30 seconds;    -   (8) Repeat steps 6 and 7 thirty-seven (37) times;    -   (9) 72° C. for 5 minutes.

In the first cycle of PCR, the annealing temperature was about themelting temperature of the 3′ annealing region of the second primers,which was 37° C. The annealing temperature in the second cycle of PCRwas about the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the first primer, which was 57° C. The annealingtemperature in the third cycle of PCR was about the melting temperatureof the entire sequence of the second primer, which was 64° C. Theannealing temperature for the remaining cycles was 64° C. Escalating theannealing temperature from TM1 to TM2 to TM3 in the first three cyclesof PCR greatly improves specificity. These annealing temperatures arerepresentative, and the skilled artisan will understand the annealingtemperatures for each cycle are dependent on the specific primers used.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results.

Purification of Fragment of Interest

The PCR products were separated from the genomic template DNA. Each PCRreaction was split into two samples and transferred to two separatewells of a Streptawell, transparent, High-Bind plate from RocheDiagnostics GmbH (catalog number 1 645 692, as listed in Roche MolecularBiochemicals, 2001 Biochemicals Catalog). For each PCR reaction, therewere two replicates, each in a separate well of a microtiter plate. Thefirst primer contained a 5′ biotin tag so the PCR products bound to theStreptavidin coated wells while the genomic template DNA did not. Thestreptavidin binding reaction was performed using a Thermomixer(Eppendorf) at 1000 rpm for 20 min. at 37° C. Each well was aspirated toremove unbound material, and washed three times with 1×PBS, with gentlemixing (Kandpal et al., Nucl. Acids Res. 18:1789-1795 (1990); Kaneoka etal., Biotechniques 10:30-34 (1991); Green et al, Nucl. Acids Res.18:6163-6164 (1990)).

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme BsmFI, which binds to the recognition site incorporated into the PCRproducts from the second primer. The digests were performed in theStreptawells following the instructions supplied with the restrictionenzyme. After digestion, the wells were washed three times with 1×PBS toremove the cleaved fragments.

Incorporation of Labeled Nucleotide

The restriction enzyme digest with BsmF I yielded a DNA fragment with a5′ overhang, which contained the SNP site or locus of interest and a 3′recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide or nucleotides in the presence of a DNApolymerase.

As discussed in detail in Example 6, the sequence of both alleles of aSNP can be determined with one labeled nucleotide in the presence of theother unlabeled nucleotides. The following components were added to eachfill in reaction; 1 μl of fluorescently labeled ddTTP, 0.5 μl ofunlabeled ddNTPs (40 μM), which contained all nucleotides exceptthymidine, 2 μl of 10× sequenase buffer, 0.25 μl of Sequenase, and wateras needed for a 20 μl reaction. The fill in reaction was performed at40° C. for 10 min. Non-fluorescently labeled ddTTP was purchased fromFermentas Inc. (Hanover, Md.). All other labeling reagents were obtainedfrom Amersham (Thermo Sequenase Dye Terminator Cycle Sequencing CoreKit, US 79565).

After labeling, each Streptawell was rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments were then released from theStreptawells by digestion with the restriction enzyme EcoRI, accordingto the manufacturer's instructions that were supplied with the enzyme.Digestion was performed for 1 hour at 37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

The samples were loaded into the lanes of a 36 cm 5% acrylamide (urea)gel (BioWhittaker Molecular Applications, Long Ranger Run Gel Packs,catalog number 50691). The samples were electrophoresed into the gel at3000 volts for 3 min. The gel was run for 3 hours on a sequencingapparatus (Hoefer SQ3 Sequencer). The gel was removed from the apparatusand scanned on the Typhoon 9400 Variable Mode Imager. The incorporatedlabeled nucleotide was detected by fluorescence. A box was drawn aroundeach band and the intensity of the band was calculated using the Typhoon9400 Variable Mode Imager software.

As seen in FIGS. 17 A-F, two bands were seen. The lower molecular weightband corresponded to the DNA molecules filled in with ddTTP at positionone complementary to the overhang. The higher molecular weight bandcorresponded to DNA molecules filled in with ddTTP at position 3complementary to the overhang.

The experiment was performed in a blind fashion. The tubes were coded sothat it was not known what tube corresponded to what template DNA. Afterthe gels were analyzed, each tube was grouped into the followingcategories: normal template DNA, Down's syndrome template DNA, 3:1mixture of Down's syndrome template DNA to normal DNA, 1:1 mixture ofnormal template DNA to Down's syndrome template DNA, 1:2.3 mixture ofDown's syndrome template DNA to normal template DNA, and 1:4 mixture ofDown's syndrome template DNA to normal template DNA. Each replicate ofeach PCR reaction successfully was grouped into the appropriatecategory, which demonstrates that the method can be used to detectabnormal DNA even if it represents only a small percentage of the totalDNA.

The percentage of allele 2 to allele 1 for each replicate of the threePCR reactions from normal template DNA are displayed in Table XVIII(also see FIG. 17A). The average percentage of allele 2 to allele 1 wascalculated by dividing the value of allele 2 by the sum of the valuesfor allele 1 and allele 2 (allele 2/(allele 1+allele 2)), which resultedin an average of 0.50025 with a standard deviation of 0.002897. Thus,allele 1 and allele 2 were present in a ratio of 50:50. While theintensity of the bands varied from one PCR reaction to another (comparereaction 1 with reaction 3), there was no difference in intensity withina PCR reaction. Furthermore, the values obtained for the two replicatesof the PCR reactions were very similar. Most of the variation wasbetween PCR reactions and was likely attributable to pipetting errors.

The percentage of allele 2 to allele 1 for each replicate of the threePCR reactions from Down's syndrome template DNA are displayed in TableXVIII (see FIG. 17B). The percentage of allele 2 to allele 1 wascalculated by dividing the value of allele 2 by the sum of the valuesfor allele 1 and allele 2 (allele 2/allele 1+allele 2), which resultedin an average of 0.301314 with a standard deviation of 0.012917. It isclear even upon analysis of the gel by the naked eye that allele 1 ispresent in a higher copy number than allele 2 (see FIG. 17B). Again,most of the variation occurs between PCR reactions and not within thereplicate of a PCR reaction. The majority of the statistical variationlikely resulted from pipetting errors.

Analysis of a single SNP was sufficient to detect the presence of thechromosomal abnormality. One SNP is sufficient provided that the “p”value of the SNP is known and that there are an adequate number ofgenomes so that statistical sampling error is not introduced into theanalysis. In this experiment, there were approximately 5,000 genomes ineach reaction.

The reactions that consisted of a mixture of Down's syndrome templateDNA to normal template DNA at a ratio of 3:1 were clearlydistinguishable from the normal template DNA, and the other mixtures ofDNA (see FIG. 17C). The calculated percentage of allele 2 to allele 1was 0.319089 with a standard deviation of 0.004346 (see Table XVIII).Likewise, the reactions that consisted of a mixture of Down's syndrometemplate DNA to normal template DNA at ratios of 1:1, and 1:2.3 weredistinguishable (see FIGS. 17D and 17E) and the values werestatistically significant from all other reactions (see Table XVIII).

As the amount of normal template DNA increased, the percentage of allele2 to allele 1 increased. With a mixture of Down's syndrome template DNAto normal template DNA of 1:4, the percentage of allele 2 to allele 1was 0397642, with a standard deviation of 0.001903 (see FIG. 17F). Thedifference between this value and the value obtained from normaltemplate DNA is statistically significant. Thus, the methods describedherein allow the detection of a chromosomal abnormality even when thesample is not a homogeneous sample of abnormal DNA.

As described above, the presence of a small fraction of DNA with anabnormal copy number of chromosomes can be detected even among a largepresence of normal DNA. It was clear, even by the naked eye, that as theamount of normal DNA increased and the amount of Down's syndrome DNAdecreased, the intensities of the bands that corresponded to alleles 1and 2 equalized.

The above example analyzed a SNP located on chromosome 21. However, anySNP may be analyzed on any chromosome including but not limited to humanchromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, X, and Y and fetal chromosomes 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, and Y. Inaddition, chromosomes from non-human organisms can be analyzed using theabove methods. Any combination of chromosomes can be analyzed. In theabove example, an extra copy of a chromosome was detected. However, thesame methods can be used to detect monosomies.

TABLE XVIII Percentage of allele 2 to allele 1 at SNP TSC0108992 usingnormal template DNA and Down's syndrome template DNA. Allele 1 Allele 22/(2 + 1) Normal Template DNA 1A 2602115 2604525 0.500231 1B 28558462923860 0.505884 2A 1954765 1941929 0.498353 2B 2084476 2068106 0.4980293A 2044147 2035719 0.498967 3B 1760291 1760543 0.500036 Mean 0.50025 STD0.002897 Down's Syndrome 1A 4046926 1595581 0.282779 1B 4275341 17362600.288818 2A 2875698 1299509 0.311244 2B 2453615 1069635 0.303593 3A3169338 1426643 0.310411 3B 3737440 1687286 0.311036 Mean 0.301314 STD0.012917 3:1 (Down's:Normal) 1A 4067623 1980770 0.327487 1B 40585061899853 0.318855 2A 2315044 1085860 0.319286 2B 2686984 1243406 0.3163573A 3880385 1790764 0.315767 3B 3718661 1724189 0.316781 Mean 0.319089STD 0.004346 1:1 (Down's:Normal) 1A 3540255 1929840 0.352798 1B 40040852161443 0.350569 2A 2358009 1282132 0.35222 2B 2158132 1238377 0.3646033A 3052330 1648677 0.350707 3B 3852682 2024012 0.344413 Mean 0.352552STD 0.006618 1:2.3 (Down's:Normal) 1A 3109326 1942597 0.384526 1B3392477 2118011 0.38436 2A 2824213 1758428 0.383715 2B 2069889 12495450.376433 3A 2335128 1433016 0.380298 3B 2916772 1797965 0.38135 Mean0.38178 STD 0.003128 1:4 (Down's:Normal) 1A 3066524 2039636 0.399446 1B3068284 2038770 0.399207 2A 2325477 1542526 0.398791 2B 2366122 15622180.397679 3A 2151205 1403120 0.394764 3B 2397046 1571360 0.395968 Mean0.397642 STD 0.001903

Example 12

As discussed above in Example 9, the ratio for allele 1 to allele 2 at aheterozygous SNP is constant. However, one factor that can influence theratio of allele 1 to allele 2 at a heterozygous SNP is a low number ofgenomes. For example, if there are 40 genomes, which means that thereare a total of 40 chromosomes of allele 1 and 40 chromosomes of allele2, it is statistically possible that the primers may anneal to 40 of thechromosomes with allele 1 but only 30 of the chromosomes with allele 2.This will affect the ratio of allele 1 to allele 2, and can erroneouslyinfluence the “p” value for a particular SNP.

Typically, whole genomic amplification, which employs degenerateoligonucleotide PCR, is used to increase low quantities of genomic DNAsamples. Oligonucleotides of 8, 10, 12, or 14 bases are used to amplifythe genome. It is thought that the primers anneal randomly throughoutthe genome, and will amplify a small genomic DNA sample intohundreds-fold more DNA for genetic analysis.

The methods described herein exploit the fact that typically the wholegenome is not of interest. Particular loci of interest located on onechromosome, or on multiple chromosomes or on chromosomes that representthe entire genome are selected for analysis. Even if the loci ofinterest are located on chromosomes for the entire genome, it ispreferential to amplify the region of those chromosomes that contain theloci of interest.

To overcome the limit of a low number of genomes, which is often seenwith fetal DNA obtained from the plasma of a pregnant female, amultiplex method can be used to increase the number of genomes. Themethod described below preferentially amplifies the chromosome orchromosomes that contain the loci of interest.

Preparation of Template DNA

A 9 ml blood sample was collected into a sterile tube from a humanvolunteer after informed consent had been granted. (Fischer Scientific,9 ml EDTA Vacuette tubes, catalog number NC9897284). The tubes were spunat 1000 rpm for ten minutes. The supernatant (the plasma) of each samplewas removed, and one milliliter of the remaining blood sample, which iscommonly referred to as the “buffy-coat” was transferred to a new tube.One milliliter of 1×PBS was added to each sample. Template DNA wasisolated using the QIAmp DNA Blood Midi Kit supplied by QIAGEN (Catalognumber 51183).

Design of Multiplex Primers

Primers were designed to anneal at various regions on chromosome 21 toincrease the copy number of the loci of interest located on chromosome21. The primers were 12 bases in length. However, primers of any lengthcan be used including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36-45, 46-55, 56-65, 66-75, 76-85, 86-95,96-105, 106-115, 116-125, and greater than 125 bases. Primers weredesigned to anneal to both the sense strand and the antisense strand.

Nine SNPs located on chromosome 21 were analyzed: TSC0397235,TSC0470003, TSC1649726, TSC1261039, TSC0310507, TSC1650432, TSC1335008,TSC0128307, and TSC0259757. Any number of SNPs can be analyzed includingbut not limited to 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70,71-80, 81-90, 91-100, 101-200, 201-300, 301-400, 401-500, 501-600,601-700, 701-800, 801-900, 901-1000, 1001-2000, 2001-3000, 3001-4000,4001-5000, 5001-6000, 6001-7000, 7001-8000, 8001-9000, 9001-10,000 andgreater than 10,000.

For each of the 9 SNPs, a 12 base primer was designed to annealapproximately 130 bases upstream of the loci of interest, and a 12 baseprimer was designed to anneal approximately 130 bases downstream of theloci of interest (herein referred to as the multiplex primers). Themultiplex primers can be designed to anneal at any distance from theloci of interest including but not limited to 10-20, 21-30, 31-40,41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130,131-140, 141-150, 151-160, 161-170, 171-180, 181-190, 191-200, 201-210,211-220, 221-230, 231-240, 241-250, 251-260, 261-270, 271-280, 281-290,291-300, 301-310, 311-320, 321-330, 331-340, 341-350, 351-360, 361-370,371-380, 381-390, 391-400, 401-410, 411-420, 421-430, 431-440, 441-450,451-460, 461-470, 471-480, 481-490, 491-500, 501-600, 601-700, 701-800,801-900, 901-1000, 1001-2000, 2001-3000, 3001-4000, 4001-5000, andgreater than 5000 bases. In addition, more than one set of multiplexprimers can be used for one SNP including but not limited to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 10-20, 21-30, 31-40, 41-50, and greater than 50.

In addition, 91 sets of forward and reverse primers were used to amplifyother regions of chromosome 21, for a total of 100 sets of primers (200primers in the reaction). These 91 primer sets were used to demonstratethat a large number of primers can be used in a single reaction withoutproducing a large number of non-specific bands. Any number of primerscan be used in the reaction including but not limited to 1-10, 11-20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-200,201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, 901-1000,1001-2000, 2001-3000, 3001-4000, 4001-5000, 5001-6000, 6001-7000,7001-8000, 8001-9000, 9001-10,000, 10,001-20,000, 20,001-30,000 andgreater than 30,000.

The multiplex primers were designed to have the same nucleotides at the3′ end of the primer. In this case, the multiplex primers ended in “AA,”wherein A indicates adenine. The primers were designed in this manner tominimize primer-dimer formation. However, the primers can terminate inany nucleotides including but not limited to adenine, guanine, cytosine,thymidine, any combination of adenine and guanine, any combination ofadenine and cytosine, any combination of adenine and thymidine, anycombination of guanine and cytosine, any combination of guanine andthymidine, or any combination of cytosine and thymidine. In addition themultiplex primers can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10 of the same nucleotides at the 3′ end.

The multiplex primers for SNP TSC0397235 were:

Forward Primer: 5′ CAAGTGTCCTAA 3′ (SEQ ID NO: 295) Reverse primer:5′ CAGCTGCTAGAA 3′ (SEQ ID NO: 296)

The multiplex primers for SNP TSC0470003 were:

Forward Primer: 5′ GGTTGAGGGCAA 3′ (SEQ ID NO: 297) Reverse primer:5′ CACAGCGGGTAA 3′ (SEQ ID NO: 298)

The multiplex primers for SNP TSC 1649726 were:

Forward Primer: 5′ TTGACTTTTTAA 3′ (SEQ ID NO: 299) Reverse primer:5′ ACAGAATGGGAA 3′ (SEQ ID NO: 300)

The multiplex primers for SNP TSC1261039 were:

Forward Primer: 5′ TGCAGGTCACAA 3′ (SEQ ID NO: 301) Reverse primer:5′ TTCTTCTTATAA 3′ (SEQ ID NO: 302)

The multiplex primers for SNP TSC0310507 were:

Forward Primer: 5′ AGGACAACCTAA 3′ (SEQ ID NO: 303) Reverse primer:5′ TGGTGTTCAGAA 3′ (SEQ ID NO: 304)

The multiplex primers for SNP TSC1650432 were:

Forward Primer: 5′ TCAGCATATGAA 3′ (SEQ ID NO: 305) Reverse primer:5′ GTTGCCACACAA 3′ (SEQ ID NO: 306)

The multiplex primers for SNP TSC1335008 were:

Forward Primer: 5′ CCCAGCTAGCAA 3′ (SEQ ID NO: 307) Reverse primer:5′ GGGTCACTGTAA 3′ (SEQ ID NO: 308)

The multiplex primers for SNP TSC0128307 were:

Forward Primer: 5′ TTAAATACCCAA 3′ (SEQ ID NO: 309) Reverse primer:5′ TTAGGAGGTTAA 3′ (SEQ ID NO: 310)

The multiplex primers for SNP TSC0259757 were:

Forward Primer: 5′ ACACAGAATCAA 3′ (SEQ ID NO: 311) Reverse primer:5′ CGCTGAGGTCAA 3′ (SEQ ID NO: 312)

Ninety-one (91) additional sets of primers, which annealed to variousregions along chromosome 21, were included in the reaction:

Set 1: Forward Primer: 5′ AAGTAGAGTCAA 3′ (SEQ ID NO: 313) Reverseprimer: 5′ CTTCCCATGGAA 3′ (SEQ ID NO: 314) Set 2: Forward Primer:5′ TTGGTTATTAAA 3′ (SEQ ID NO: 315) Reverse primer: 5′ CAACTTACTGAA 3′(SEQ ID NO: 316) Set 3: Forward Primer: 5′ CACTAAGTGAAA 3′ (SEQ ID NO:317) Reverse primer: 5′ CTCACCTGCCAA 3′ (SEQ ID NO: 318) Set 4: ForwardPrimer: 5′ ATGCATATATAA 3′ (SEQ ID NO: 319) Reverse primer:5′ AGAGATCAGCAA 3′ (SEQ ID NO: 320) Set 5: Forward Primer:5′ TATATTTTTCAA 3′ (SEQ ID NO: 321) Reverse primer: 5′ CAGAAAGCAGAA 3′(SEQ ID NO: 322) Set 6: Forward Primer: 5′ GTATTGGGTTAA 3′ (SEQ ID NO:323) Reverse primer: 5′ CTGACCCAGGAA 3′ (SEQ ID NO: 324) Set 7: ForwardPrimer: 5′ CAGTTTTCCCAA 3′ (SEQ ID NO: 325) Reverse primer:5′ AGGGCACAGGAA 3′ (SEQ ID NO: 326) Set 8: Forward Primer:5′ GTATCAGAGGAA 3′ (SEQ ID NO: 327) Reverse primer: 5′ GCATGAAAAGAA 3′(SEQ ID NO: 328) Set 9: Forward Primer: 5′ GATTTGACAGAA 3′ (SEQ ID NO:329) Reverse primer: 5′ TACAGTTTACAA 3′ (SEQ ID NO: 330) Set 10: ForwardPrimer: 5′ TGTGATTTTTAA 3′ (SEQ ID NO: 331) Reverse primer:5′ TTATGTTCTCAA 3′ (SEQ ID NO: 332) Set 11: Forward Primer:5′ CAAGTACTTGAA 3′ (SEQ ID NO: 333) Reverse primer: 5′ CTTGTGTGGCAA 3′(SEQ ID NO: 334) Set 12: Forward Primer: 5′ AGACTTCTGCAA 3′ (SEQ ID NO:335) Reverse primer: 5′ GTTGTCTTTCAA 3′ (SEQ ID NO: 336) Set 13: ForwardPrimer: 5′ GGGACACTCCAA 3′ (SEQ ID NO: 337) Reverse primer:5′ ATTATTATTCAA 3′ (SEQ ID NO: 338) Set 14: Forward Primer:5′ ACATGATGACAA 3′ (SEQ ID NO: 339) Reverse primer: 5′ TCAATTATAGAA 3′(SEQ ID NO: 340) Set 15: Forward Primer: 5′ CTATGGGCTGAA 3′ (SEQ ID NO:341) Reverse primer: 5′ TGTGTGCCTGAA 3′ (SEQ ID NO: 342) Set 16: ForwardPrimer: 5′ CCATTTGTTGAA 3′ (SEQ ID NO: 343) Reverse primer:5′ TCTCCATCAAAA 3′ (SEQ ID NO: 344) Set 17: Forward Primer:5′ AATGCTGACAAA 3′ (SEQ ID NO: 345) Reverse primer: 5′ TTTCATGTCCAA 3′(SEQ ID NO: 346) Set 18: Forward Primer: 5′ GGCCTCTTGGAA 3′ (SEQ ID NO:347) Reverse primer: 5′ TCATTTTTTGAA 3′ (SEQ ID NO: 348) Set 19: ForwardPrimer: 5′ GGACTACCATAA 3′ (SEQ ID NO: 349) Reverse primer:5′ AGTCACTCAGAA 3′ (SEQ ID NO: 350) Set 20: Forward Primer:5′ CCTTGGCAGGAA 3′ (SEQ ID NO: 351) Reverse primer: 5′ TTTCTGGTAGAA 3′(SEQ ID NO: 352) Set 21: Forward Primer: 5′ CCCCCCCCCGAA 3′ (SEQ ID NO:353) Reverse primer: 5′ GCCCAGGCAGAA 3′ (SEQ ID NO: 354) Set 22: ForwardPrimer: 5′ GAATGCGAAGAA 3′ (SEQ ID NO: 355) Reverse primer:5′ TTAGGTAGAGAA 3′ (SEQ ID NO: 356) Set 23: Forward Primer:5′ TGCTTTGGTCAA 3′ (SEQ ID NO: 357) Reverse primer: 5′ GCCCATTAATAA 3′(SEQ ID NO: 358) Set 24: Forward Primer: 5′ TGAGATCTTTAA 3′ (SEQ ID NO:359) Reverse primer: 5′ CAGTTTGTTCAA 3′ (SEQ ID NO: 360) Set 25: ForwardPrimer: 5′ GCTGGGCAAGAA 3′ (SEQ ID NO: 361) Reverse primer:5′ AGTCAAAGTCAA 3′ (SEQ ID NO: 362) Set 26: Forward Primer:5′ TCTCTGCAGTAA 3′ (SEQ ID NO: 363) Reverse primer: 5′ TGAATAACTTAA 3′(SEQ ID NO: 364) Set 27: Forward Primer: 5′ CGGTTAGAAAAA 3′ (SEQ ID NO:365) Reverse primer: 5′ CATCCCTTTCAA 3′ (SEQ ID NO: 366) Set 28: ForwardPrimer: 5′ TCTCTTTCTGAA 3′ (SEQ ID NO: 367) Reverse primer:5′ CTCAGATTGTAA 3′ (SEQ ID NO: 368) Set 29: Forward Primer:5′ TTTGCACCAGAA 3′ (SEQ ID NO: 369) Reverse primer: 5′ GGTTAACATGAA 3′(SEQ ID NO: 370) Set 30: Forward Primer: 5′ ATTATCAACTAA 3′ (SEQ ID NO:371) Reverse primer: 5′ GCCATTTTGTAA 3′ (SEQ ID NO: 372) Set 31: ForwardPrimer: 5′ GATCTAGATGAA 3′ (SEQ ID NO: 373) Reverse primer:5′ TTAATGTATTAA 3′ (SEQ ID NO: 374) Set 32: Forward Primer:5′ CTAGGGAGACAA 3′ (SEQ ID NO: 375) Reverse primer: 5′ TGGAGGAGACAA 3′(SEQ ID NO: 376) Set 33: Forward Primer: 5′ CATCACATTTAA 3′ (SEQ ID NO:377) Reverse primer: 5′ GGGGTCCTGCAA 3′ (SEQ ID NO: 378) Set 34: ForwardPrimer: 5′ CAGTTGTGCTAA 3′ (SEQ ID NO: 379) Reverse primer:5′ TCTGCAGCCTAA 3′ (SEQ ID NO: 380) Set 35: Forward Primer:5′ GAGTCATTTAAA 3′ (SEQ ID NO: 381) Reverse primer: 5′ TCTATGGATTAA 3′(SEQ ID NO: 382) Set 36: Forward Primer: 5′ CAAAAAGTAGAA 3′ (SEQ ID NO:383) Reverse primer: 5′ AATATACTCCAA 3′ (SEQ ID NO: 384) Set 37: ForwardPrimer: 5′ CGTCCAGCACAA 3′ (SEQ ID NO: 385) Reverse primer:5′ GGATGGTGAGAA 3′ (SEQ ID NO: 386) Set 38: Forward Primer:5′ TCTCCTTTGTAA 3′ (SEQ ID NO: 387) Reverse primer: 5′ TCGTTATTTCAA 3′(SEQ ID NO: 388) Set 39: Forward Primer: 5′ GATTTTATAGAA 3′ (SEQ ID NO:389) Reverse primer: 5′ AGACATAAGCAA 3′ (SEQ ID NO: 390) Set 40: ForwardPrimer: 5′ TTCACCTCACAA 3′ (SEQ ID NO: 391) Reverse primer:5′ GGATTGCTTGAA 3′ (SEQ ID NO: 392) Set 41: Forward Primer:5′ ACTGCATGTGAA 3′ (SEQ ID NO: 393) Reverse primer: 5′ TTTATCACAGAA 3′(SEQ ID NO: 394) Set 42: Forward Primer: 5′ TCAGTAACACAA 3′ (SEQ ID NO:395) Reverse primer: 5′ TACATCTTTGAA 3′ (SEQ ID NO: 396) Set 43: ForwardPrimer: 5′ TTGTTTCAGTAA 3′ (SEQ ID NO: 397) Reverse primer:5′ TATGAGCATCAA 3′ (SEQ ID NO: 398) Set 44: Forward Primer:5′ CTCAGCAGGCAA 3′ (SEQ ID NO: 399) Reverse primer: 5′ ACCCCTGTATAA 3′(SEQ ID NO: 400) Set 45: Forward Primer: 5′ TCTGCTCAGCAA 3′ (SEQ ID NO:401) Reverse primer: 5′ GTTCTTTTTTAA 3′ (SEQ ID NO: 402) Set 46: ForwardPrimer: 5′ GTGATAATCCAA 3′ (SEQ ID NO: 403) Reverse primer:5′ GAGCCCTCAGAA 3′ (SEQ ID NO: 404) Set 47: Forward Primer:5′ TTTATTGGTTAA 3′ (SEQ ID NO: 405) Reverse primer: 5′ GGTACTGGGCAA 3′(SEQ ID NO: 406) Set 48: Forward Primer: 5′ AGTGTTTTTCAA 3′ (SEQ ID NO:407) Reverse primer: 5′ TGTTATTGGTAA 3′ (SEQ ID NO: 408) Set 49: ForwardPrimer: 5′ GCGCATTCACAA 3′ (SEQ ID NO: 409) Reverse primer:5′ AAACAAAAGCAA 3′ (SEQ ID NO: 410) Set 50: Forward Primer:5′ TATATGATAGAA 3′ (SEQ ID NO: 411) Reverse primer: 5′ TCCCAGTTCCAA 3′(SEQ ID NO: 412) Set 51: Forward Primer: 5′ AAAGCCCATAAA 3′ (SEQ ID NO:413) Reverse primer: 5′ TGTCATCCACAA 3′ (SEQ ID NO: 414) Set 52: ForwardPrimer: 5′ TTGTGAATGCAA 3′ (SEQ ID NO: 415) Reverse primer:5′ GTATTCATACAA 3′ (SEQ ID NO: 416) Set 53: Forward Primer:5′ TGACATAGGGAA 3′ (SEQ ID NO: 417) Reverse primer: 5′ AGCAAATTGCAA 3′(SEQ ID NO: 418) Set 54: Forward Primer: 5′ AGTAGATGTTAA 3′ (SEQ ID NO:419) Reverse primer: 5′ AAAAGATAATAA 3′ (SEQ ID NO: 420) Set 55: ForwardPrimer: 5′ ACCTCATGGGAA 3′ (SEQ ID NO: 421) Reverse primer:5′ TGGTCGACCTAA 3′ (SEQ ID NO: 422) Set 56: Forward Primer:5′ TTTGCATGGTAA 3′ (SEQ ID NO: 423) Reverse primer: 5′ GCGGCTGCCGAA 3′(SEQ ID NO: 424) Set 57: Forward Primer: 5′ TCAGGAGTCTAA 3′ (SEQ ID NO:425) Reverse primer: 5′ GCCTACCAGGAA 3′ (SEQ ID NO: 426) Set 58: ForwardPrimer: 5′ ATCTTCTGTTAA 3′ (SEQ ID NO: 427) Reverse primer:5′ AGGTAAGGACAA 3′ (SEQ ID NO: 428) Set 59: Forward Primer:5′ TGCTTTGAGGAA 3′ (SEQ ID NO: 429) Reverse primer: 5′ AACAGTTTTAAA 3′(SEQ ID NO: 430) Set 60: Forward Primer: 5′ TTAAATGTTTAA 3′ (SEQ ID NO:431) Reverse primer: 5′ ATAGAAAATCAA 3′ (SEQ ID NO: 432) Set 61: ForwardPrimer: 5′ GTGTTGTGTTAA 3′ (SEQ ID NO: 433) Reverse primer:5′ GAGGACCTCGAA 3′ (SEQ ID NO: 434) Set 62: Forward Primer:5′ AGAGGCTGAGAA 3′ (SEQ ID NO: 435) Reverse primer: 5′ GGTATTTATTAA 3′(SEQ ID NO: 436) Set 63: Forward Primer: 5′ ATTTATCTGGAA 3′ (SEQ ID NO:437) Reverse primer: 5′ AGTGCAAACTAA 3′ (SEQ ID NO: 438) Set 64: ForwardPrimer: 5′ TGAACACCTTAA 3′ (SEQ ID NO: 439) Reverse primer:5′ AATTTTTTCTAA 3′ (SEQ ID NO: 440) Set 65: Forward Primer:5′ TTACTATTATAA 3′ (SEQ ID NO: 441) Reverse primer: 5′ TGCTATAGTGAA 3′(SEQ ID NO: 442) Set 66: Forward Primer: 5′ TGGACTATGGAA 3′ (SEQ ID NO:443) Reverse primer: 5′ CTGCAGTCCGAA 3′ (SEQ ID NO: 444) Set 67: ForwardPrimer: 5′ GCTACTGCCCAA 3′ (SEQ ID NO: 445) Reverse primer:5′ TCACATGGTGAA 3′ (SEQ ID NO: 446) Set 68: Forward Primer:5′ GTGGCTCTGGAA 3′ (SEQ ID NO: 447) Reverse primer: 5′ GAATTCCATTAA 3′(SEQ ID NO: 448) Set 69: Forward Primer: 5′ TGGGGTGTCCAA 3′ (SEQ ID NO:449) Reverse primer: 5′ GCAAGCTCCGAA 3′ (SEQ ID NO: 450) Set 70: ForwardPrimer: 5′ ATGTTTTTTCAA 3′ (SEQ ID NO: 451) Reverse primer:5′ AGATCTGTTGAA 3′ (SEQ ID NO: 452) Set 71: Forward Primer:5′ AAGTGCTGTGAA 3′ (SEQ ID NO: 453) Reverse primer: 5′ ACTTTTTTGGAA 3′(SEQ ID NO: 454) Set 72: Forward Primer: 5′ AATCGGCAGGAA 3′ (SEQ ID NO:455) Reverse primer: 5′ GGCATGTCACAA 3′ (SEQ ID NO: 456) Set 73: ForwardPrimer: 5′ AGGAAGAAAGAA 3′ (SEQ ID NO: 457) Reverse primer:5′ CAGTTTCACCAA 3′ (SEQ ID NO: 458) Set 74: Forward Primer:5′ CACAGAATTTAA 3′ (SEQ ID NO: 459) Reverse primer: 5′ AAGAATAAGTAA 3′(SEQ ID NO: 460) Set 75: Forward Primer: 5′ GGGATAGTACAA 3′ (SEQ ID NO:461) Reverse primer: 5′ TTCCCATGATAA 3′ (SEQ ID NO: 462) Set 76: ForwardPrimer: 5′ TGATTAGTTGAA 3′ (SEQ ID NO: 463) Reverse primer:5′ GCATTCAGTGAA 3′ (SEQ ID NO: 464) Set 77: Forward Primer:5′ AGGGAATATTAA 3′ (SEQ ID NO: 465) Reverse primer: 5′ GACCTTAGGTAA 3′(SEQ ID NO: 466) Set 78: Forward Primer: 5′ TTCTTTTCACAA 3′ (SEQ ID NO:467) Reverse primer: 5′ CCAAACTAAGAA 3′ (SEQ ID NO: 468) Set 79: ForwardPrimer: 5′ GTGCTCTTAGAA 3′ (SEQ ID NO: 469) Reverse primer:5′ ATGAGTTTAGAA 3′ (SEQ ID NO: 470) Set 80: Forward Primer:5′ ATGAGCATAGAA 3′ (SEQ ID NO: 471) Reverse primer: 5′ GACAAATGAGAA 3′(SEQ ID NO: 472) Set 81: Forward Primer: 5′ AAACCCAGAGAA 3′ (SEQ ID NO:473) Reverse primer: 5′ CCTCACACAGAA 3′ (SEQ ID NO: 474) Set 82: ForwardPrimer: 5′ CACACTGTGGAA 3′ (SEQ ID NO: 475) Reverse primer:5′ CACTGTACCCAA 3′ (SEQ ID NO: 476) Set 83: Forward Primer:5′ GTAGTATTTCAA 3′ (SEQ ID NO: 477) Reverse primer: 5′ TGGATACACTAA 3′(SEQ ID NO: 478) Set 84: Forward Primer: 5′ CCCATGATTCAA 3′ (SEQ ID NO:479) Reverse primer: 5′ TCATAGGAGGAA 3′ (SEQ ID NO: 480) Set 85: ForwardPrimer: 5′ AGGAAAGAGAAA 3′ (SEQ ID NO: 481) Reverse primer:5′ ATATGGTGATAA 3′ (SEQ ID NO: 482) Set 86: Forward Primer:5′ GATGCCATCCAA 3′ (SEQ ID NO: 483) Reverse primer: 5′ ATACTATTTCAA 3′(SEQ ID NO: 484) Set 87: Forward Primer: 5′ GTGTGCATGGAA 3′ (SEQ ID NO:485) Reverse primer: 5′ AGGTGTTGAGAA 3′ (SEQ ID NO: 486) Set 88: ForwardPrimer: 5′ CAGCCTGGGCAA 3′ (SEQ ID NO: 487) Reverse primer:5′ GGAGCTCTACAA 3′ (SEQ ID NO: 488) Set 89: Forward Primer:5′ AACTAAGGTTAA 3′ (SEQ ID NO: 489) Reverse primer: 5′ AACTTATGTTAA 3′(SEQ ID NO: 490) Set 90: Forward Primer: 5′ ATCTCAACAGAA 3′ (SEQ ID NO:491) Reverse primer: 5′ TAACAATGTGAA 3′ (SEQ ID NO: 492) Set 91: ForwardPrimer 5′ AAGGATCAGGAA 3′ (SEQ ID NO: 493) Reverse primer:5′ CTCAAGTCTTAA 3′ (SEQ ID NO: 494)

Multiplex PCR

Regions on chromosome 21 surrounding SNPs TSC0397235, TSC0470003,TSC1649726, TSC1261039, TSC0310507, TSC1650432, TSC1335008, TSC0128307,and TSC0259757 were amplified from the template genomic DNA using thepolymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202,incorporated herein by reference). This PCR reaction used primers thatannealed approximately 130 bases upstream and downstream of the loci ofinterest. It was used to increases the number of copies of the loci ofinterest to eliminate any errors that may result from a low number ofgenomes.

For increased specificity, a “hot-start” PCR reaction was used. PCRreactions were performed using the HotStarTaq Master Mix Kit supplied byQIAGEN (catalog number 203443). The amount of template DNA and primerper reaction can be optimized for each locus of interest. In thisexample, 15 ng of template human genomic DNA and 5 μM of each primerwere used.

Two microliters of each forward and reverse primer, at concentrations of5 mM were pooled into a single microcentrifuge tube and mixed. Eightmicroliters of the primer mix was used in a total PCR reaction volume of40 μl (1.5 μl of template DNA, 10.5 μl of sterile water, 8 μl of primermix, and 20 μl of HotStar Taq). Twenty-five cycles of PCR wereperformed. The following PCR conditions were used:

-   -   (1) 95° C. for 15 minutes;    -   (2) 95° C. for 30 seconds;    -   (3) 4° C. for 30 seconds;    -   (4) 37° C. for 30 seconds;    -   (5) Repeat steps 2-4 twenty-four (24) times;    -   (6) 72° C. for 10 minutes.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results.

In another embodiment, the loci of interest are amplified using 6-baseoligonucleotides, 7-base oligonucleotides, 8-base oligonucleotides,9-base oligonucleotides, 10-base oligonucleotides, 11-baseoligonucleotides, 12-base oligonucleotides, 13-base oligonucleotides,14-base oligonucleotides, or greater than 14-base oligonucleotides. In apreferred embodiment, 6-base oligonucleotides, 7-base oligonucleotides,8-base oligonucleotides, 9-base oligonucleotides, 10-baseoligonucleotides, 11-base oligonucleotides, or 12-base oligonucleotidesare used to amplify the loci of interest. In another embodiment, anynumber of oligonucleotides can be used including but not limited to 1-5,5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-100,100-500, 500-1000, 1000-2000, 2000-4000, 4000-8000, 8000-10,000 orgreater than 10,000. With a small number of random oligos, theconcentration of the oligos is large enough to allow efficientamplification, and yet, the number of oligos is small enough that itdoes not cause interference between the oligos. This allows efficientamplification of the genome.

In another embodiment, the upstream and downstream sequences of the lociof interest are analyzed to identify a 6-base, 7-base, 8-base, 9-base,10-base, 11-base, or 12-base sequence that is present in the sequenceupstream or downstream for each of the loci of interest, which is thenused to amplify the loci of interest. In another embodiment, any numberof 6-base oligonucleotides can be used to amplify the loci of interestincluding but not limited to 1-10, 10-50, 50-100, 100-200, 200-500, orgreater than 500.

In another embodiment, the number of loci of interest from a smallnumber of genomes can be increased by amplifying a limited number of theloci of interest, followed by removal of the primers, and amplificationof the remaining loci of interest. All the loci of interest do not haveto be multiplexed in one reaction. Any number of experimentallydetermined loci of interest can be multiplexed in a single reactionincluding but not limited to 1-5, 5-10, 10-25, 25-50, 50-100, 100-200,200-400, or greater than 400. After increasing the number of copies ofthese loci of interest, the sample can be passed through a column thatallows the amplified products to bind and the primers and unused dNTPsto be removed. After eluting the bound products from the column,different loci of interest can be amplified in a single reaction. Thisreduces the amount of interaction between the primers.

Other methods of genomic amplification can also be used to increase thecopy number of the loci of interest including but not limited to primerextension preamplification (PEP) (Zhang et al., PNAS, 89:5847-51, 1992),degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius, et al.,Genomics 13:718-25, 1992), strand displacement amplification using DNApolymerase from bacteriophage 29, which undergoes rolling circlereplication (Dean et al., Genomic Research 11:1095-99, 2001), multipledisplacement amplification (U.S. Pat. No. 6,124,120), REPLI-g™ WholeGenome Amplification kits, and Tagged PCR.

Purification of Fragment of Interest

The excess primers and nucleotides were removed from the reaction byusing Qiagen MinElute PCR purification kits (Qiagen, Catalog Number28004). The reactions were performed following the manufacturer'sinstructions supplied with the columns. The DNA was eluted in 100 μl ofsterile water.

PCR Reaction Two

SNP TSC0397235 was amplified using the following primer set:

First Primer: (SEQ ID NO: 495) 5′ TTAGTCATCGCAGAATTCTACTTCTTTCTGAAGTGGGA3′ Second primer: (SEQ ID NO: 496) 5′ GGACAGCTCGATGGGACTAATGCATACTC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI, and was designed to anneal 103bases from the locus of interest. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC0470003 was amplified using the following primer set:

First Primer: (SEQ ID NO: 497) 5′ GTAGCCACTGGTGAATTCGTGCCATCGCAAAAGAATAA3′ Second primer: (SEQ ID NO: 498) 5′ ATTAGAATGATGGGGACCCCTGTCTTCCC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI, and was designed to anneal 80bases from the locus of interest. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC1649726 was amplified using the following primer set:

First Primer: (SEQ ID NO: 499) 5′ ACGCATAGGAAGGAATTCATTCTGACACGTGTGAGATA3′ Second primer: (SEQ ID NO: 500) 5′ GAAATTGACCACGGGACTGCACACTTTTC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI, and was designed to anneal 113bases from the locus of interest. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC1261039 was amplified using the following primer set:

First Primer: (SEQ ID NO: 501) 5′ CGGTAAATCGGAGAATTCAAGTTGAGGCATGCATCCAT3′ Second primer: (SEQ ID NO: 502) 5′ TCGGGGCTCAGCGGGACCACAGCCACTCC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI, and was designed to anneal 54bases from the locus of interest. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC0310507 was amplified using the following primer set:

First Primer: (SEQ ID NO: 503) 5′ TCTATGCACCACGAATTCAATATGTGTTCAAGGACATT3′ Second primer: (SEQ ID NO: 504) 5′ TGCTTAATCGGTGGGACTTGTAATTGTAC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI, and was designed to anneal 93bases from the locus of interest. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC1650432 was amplified using the following primer set:

First Primer: (SEQ ID NO: 505) 5′ CGCGTTGTATGCGAATTCCCTGGGGTATAAAGATAAGA3′ Second primer: (SEQ ID NO: 506) 5′ CTCACGGGAACTGGGACACCTGACCCTGC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI, and was designed to anneal 80bases from the locus of interest. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC1335008 was amplified using the following primer set:

First Primer: (SEQ ID NO: 507) 5′ GTCTTGCCGCTTGAATTCCCATAGAAGAATGCGCCAAA3′ Second primer: (SEQ ID NO: 508) 5′ TTGAGTAGTACAGGGACACACTAACAGAC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI, and was designed to anneal 94bases from the locus of interest. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC0128307 was amplified using the following primer set:

First Primer: (SEQ ID NO: 509) 5′ AATACTGTAGGTGAATTCTTGCCTAAGCATTTTCCCAG3′ Second primer: (SEQ ID NO: 510) 5′ GTGTTGACATTCGGGACTGTAATCTTGAC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI, and was designed to anneal 54bases from the locus of interest. The second primer contained therecognition site for the restriction enzyme BsmF I.

SNP TSC0259757 was amplified using the following primer set:

First Primer: (SEQ ID NO: 511) 5′ TCTGTAGATTCGGAATTCTTTAGAGCCTGTGCGCTGAG3′ Second primer: (SEQ ID NO: 512) 5′ CGTACCAGTACAGGGACGCAAACTGAGAC 3′

The first primer contained a biotin tag at the 5′ end and a recognitionsite for the restriction enzyme EcoRI, and was designed to anneal 100bases from the locus of interest. The second primer contained therecognition site for the restriction enzyme BsmF I.

All loci of interest were amplified from the template genomic DNA usingthe polymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and4,683,202, incorporated herein by reference). In this example, the lociof interest were amplified in separate reaction tubes but they can alsobe amplified together in a single PCR reaction. For increasedspecificity, a “hot-start” PCR was used. PCR reactions were performedusing the HotStarTaq Master Mix Kit supplied by QIAGEN (catalog number203443).

One microliter of the elutate from the multiplex reaction (PCR producteluted from the MinElute column) was used as template DNA for each PCRreaction. Each SNP was amplified in triplicate when the multiplex samplewas used as the template. As a control, each SNP was amplified from 15ng of the original template DNA (DNA that did not undergo the multiplexreaction). The amount of template DNA and primer per reaction can beoptimized for each locus of interest but in this example, 5 μM of eachprimer was used. Forty cycles of PCR were performed. The following PCRconditions were used:

-   -   (1) 95° C. for 15 minutes and 15 seconds;    -   (2) 37° C. for 30 seconds;    -   (3) 95° C. for 30 seconds;    -   (4) 57° C. for 30 seconds;    -   (5) 95° C. for 30 seconds;    -   (6) 64° C. for 30 seconds;    -   (7) 95° C. for 30 seconds;    -   (8) Repeat steps 6 and 7 thirty nine (39) times;    -   (9) 72° C. for 5 minutes.

In the first cycle of PCR, the annealing temperature was about themelting temperature of the 3′ annealing region of the second primers,which was 37° C. The annealing temperature in the second cycle of PCRwas about the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the first primer, which was 57° C. The annealingtemperature in the third cycle of PCR was about the melting temperatureof the entire sequence of the second primer, which was 64° C. Theannealing temperature for the remaining cycles was 64° C. Escalating theannealing temperature from TM1 to TM2 to TM3 in the first three cyclesof PCR greatly improves specificity. These annealing temperatures arerepresentative, and the skilled artisan will understand the annealingtemperatures for each cycle are dependent on the specific primers used.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results.

Agarose Gel Analysis

Four microliters of a twenty microliter PCR reaction for each SNP fromthe original template DNA was analyzed by agarose gel electrophoresis(see FIG. 18A). Four microliters of a twenty microliter PCR reaction foreach SNP that was amplified from the multiplexed template was analyzedon by agarose gel electrophoresis (see FIG. 18B).

As seen in FIG. 18A, for 8/9 of the SNPs amplified from the originaltemplate DNA, a single band of high intensity was seen (lanes 1-3, and5-9). The band migrated at the correct position for each of the 8 SNPs.Amplification of TSC1261039 from the original template DNA produced aband of high intensity, which migrated at the correct position, and afaint band of lower molecular weight (lane 4). Only two bands were seen,and the bands could clearly be distinguished based on molecular weight.The PCR method described herein allows clean amplification of the lociof interest from genomic DNA without any concentration or enrichment ofthe loci of interest.

As seen in FIG. 18B, the primers used to amplify SNPs TSC0397235,TSC0470003, TSC0310507, and TSC0128307 from the multiplexed template DNAproduced a single band of high intensity, which migrated at the correctposition (lanes 1, 2, 5, and 8). No additional bands were introduceddespite the fact that the multiplex reaction contained two hundredprimers. While the multiplex primers were 12 bases in length and likelyannealed to additional sequences other than those located on chromosome21, the products were not seen because the bands were not amplified inthe second PCR reaction. The second PCR reaction employed primersspecific for the loci of interest and used asymmetric oligonucleotidesand escalating annealing temperatures, which allows specificamplification from the genome (see Example 1).

Amplification of TSC1649726 from the multiplex template DNA produced oneband of high intensity and two weaker bands, which could clearly bedistinguished based on molecular weight (see FIG. 18B, lane 3).Amplification of TSC1261039 from the multiplex template DNA produced ahigh intensity band of the correct molecular weight and a faint band oflower molecular weight (see FIG. 18B, lane 4). The low molecular weightband was the same size as the band seen from the amplification ofTSC1261039 from the original template DNA (compare FIG. 18A, lane 4 withFIG. 18B, lane 4). Thus, amplification of TSC1261039 on the multiplextemplate DNA did not introduce any additional non-specific bands

Amplification of SNPs TSC1650432, TSC1335008, and TSC0259757 from themultiplex template DNA produced one band of high intensity, whichmigrated at the correct position, and one weaker band (lanes 6, 7, and9). For SNPs TSC1650432 and TSC0259757, the weaker band was of lowermolecular weight, and clearly was distinguishable from the band ofinterest (see FIG. 18B, lanes 6 and 9). For SNP TSC1335008, the weakerband was of slightly higher molecular weight. However, the correct bandcan be identified by comparing to the amplification products ofTSC1335008 from the original template DNA, (compare FIG. 18A, lane 7 andFIG. 18B, lane 7). The PCR conditions can also be optimized forTSC1335008. All 9 SNPs were amplified under the exact same conditions,which produced clearly distinguishable bands for the amplified SNPs.

Purification of Fragment of Interest

The PCR products were separated from the genomic template DNA. One halfof the PCR reaction was transferred to a well of a Streptawell,transparent, High-Bind plate from Roche Diagnostics GmbH (catalog number1 645 692, as listed in Roche Molecular Biochemicals, 2001 BiochemicalsCatalog). The first primers contained a 5′ biotin tag so the PCRproducts bound to the Streptavidin coated wells while the genomictemplate DNA did not. The streptavidin binding reaction was performedusing a Thermomixer (Eppendorf) at 1000 rpm for 20 min. at 37° C. Eachwell was aspirated to remove unbound material, and washed three timeswith 1×PBS, with gentle mixing (Kandpal et al., Nucl. Acids Res.18:1789-1795 (1990); Kaneoka et al. Biotechniques 10:30-34 (1.991);Green et al., Nucl. Acids Res. 18:6163-6164 (1990)).

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme BsmFI, which binds to the recognition site incorporated into the PCRproducts from the second primer. The digests were performed in theStreptawells following the instructions supplied with the restrictionenzyme. After digestion, the wells were washed three times with PBS toremove the cleaved fragments.

Incorporation of Labeled Nucleotide

The restriction enzyme digest with BsmF I yielded a DNA fragment with a5′ overhang, which contained the SNIP site or locus of interest and a 3′recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide or nucleotides in the presence of a DNApolymerase.

As discussed in detail in Example 6, the sequence of both alleles of aSNP can be determined by using one labeled nucleotide in the presence ofthe other unlabeled nucleotides. The following components were added toeach fill in reaction: 1 μl of fluorescently labeled ddGTP, 0.5 μl ofunlabeled ddNTPs (40 μM), which contained all nucleotides exceptguanine, 2 μl of 10× sequenase buffer, 025 μl of Sequenase, and water asneeded for a 20 μl reaction. The fill in reaction was performed at 40°C. for 10 min. Non-fluorescently labeled ddNTP was purchased fromFermentas Inc. (Hanover, Md.). All other labeling reagents were obtainedfrom Amersham (Thermo Sequenase Dye Terminator Cycle Sequencing CoreKit, US 79565).

After labeling, each Streptawell was rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments then were released from theStreptawells by digestion with the restriction enzyme EcoRI, accordingto the manufacturer's instructions that were supplied with the enzyme.Digestion was performed for 1 hour at 37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

The samples were loaded into a lane of a 36 cm 5% acrylamide (urea) gel(BioWhittaker Molecular Applications, Long Ranger Run Gel Packs, catalognumber 50691). The samples were electrophoresed into the gel at 3000volts for 3 min. The gel was run for 3 hours on a sequencing apparatus(Hoefer SQ3 Sequencer). The gel was removed from the apparatus andscanned on the Typhoon 9400 Variable Mode Imager. The incorporatedlabeled nucleotide was detected by fluorescence. A box was drawn aroundeach band and the intensity of the band was calculated using theImageQuant software.

Below, a schematic of the 5′ overhang for TSC0470003 after digestionwith BsmF I is depicted:

5′ CTCT 3′ GAGA R A C C Overhang position 1 2 3 4

The observed nucleotides for TSC0470003 are adenine and guanine on thesense strand (herein depicted as the top strand). The third position ofthe overhang corresponds to cytosine, which is complementary to guanine.Labeled ddGTP was used in the presence of unlabeled dATP, dCTP, anddTTP. Schematics of the DNA molecules after the fill-in reaction aredepicted below:

Allele 1 5′ CTCT G* 3′ GAGA C A C C Overhang position 1 2 3 4 Allele 25′ CTCT A T G* 3′ GAGA T A C C Overhang position 1 2 3 4

Two bands were seen; the lower molecular weight band corresponded to theDNA molecules filled in with ddGTP at position 1 complementary to theoverhang and the higher molecular weight band corresponded to the DNAmolecules filled in with ddGTP at position 3 complementary to theoverhang (see FIG. 19).

The percentage of allele 2 to allele 1 at TSC0470003 after amplificationfrom the original template DNA and the multiplexed template DNA wascalculated. The use of one fluorescently labeled nucleotide to detectboth alleles in a single reaction reduces the amount of error that isintroduced through pipetting reactions, and the error that is introducedthrough the quantum coefficients of different dyes.

For SNP TSC047003, the percentage of allele 2 to allele 1 was calculatedby dividing the value of allele 2 by the sum of the values for allele 2and allele 1. The percentage of allele 2 to allele 1 for TSC047003 onthe original template DNA was calculated to be 0.539 (see Table XIX).Three PCR reactions were performed for each SNP on the multiplexedtemplate DNA. The average percentage of allele 2 to allele 1 forTSC047003 on the multiplexed DNA was 0.49 with a standard deviation of0.0319 (see Table XIX). There was no statistically significantdifference between the percentage obtained on the original template DNAand the multiplexed template DNA.

For SNP TSC1261039, the percentage of allele 2 to allele 1 forTSC1261039 on the original template DNA was calculated to be 0.44 (seeTable XIX). Three PCR reactions were performed for each SNP on themultiplexed template DNA (see FIG. 19B). The average percentage ofallele 2 to allele 1 for TSC1261039 on the multiplexed DNA was 0.468with a standard deviation of 0.05683 (see Table XIX). There was nostatistically significant difference between the percentages of allele 2to allele 1 obtained on the original template DNA and the multiplexedtemplate DNA.

The variation seen in the percentage of allele 2 to allele 1 forTSC1261039 on the multiplexed template DNA was likely due to pipettingreactions. The variation can be reduced by increasing the number ofreplicates. With a large number of replicates, a percentage can beobtained with minimum statistical variation.

Likewise, there was no statistical difference between the percentage ofallele 2 to allele 1 on the original template DNA and on the multiplexedtemplate DNA for SNPs TSC0310507 and TSC1335008 (see Table XIX, andFIGS. 19C and 19D). Thus, a multiplex reaction can be used to increasethe number of chromosomal regions containing the loci of interestwithout affecting the percentage of one allele to the other at thevariable sites.

TABLE XIX Percentage of allele 2 to allele 1 at various SNPs with andwithout multiplexing. Allele 1 Allele 2 2/(2 + 1) TSC047003 IA 55354186487873 0.539608748 M1 4804358 4886716 0.504249168 M2 5549389 59585850.517778803 M3 8356275 7030245 0.45690936 Mean (M1-M3) 0.49297911 STDEV0.031961429 TSC1261039 IA 3488765 2768066 0.442407027 M1 3603388 25732440.41660957 M2 4470423 5026872 0.529295131 M3 4306015 36694012 0.46008898Mean (M1-M3) 0.46866456 STDEV 0.056830136 TSC0310507 IA 2966511 26881900.475390299 M1 4084472 2963451 0.420471535 M2 4509891 4052892 0.47331481M3 7173191 4642069 0.39288759 Mean (M1-M3) 0.428891312 STDEV 0.040869352TSC1335008 IA 2311629 2553016 0.524810341 M1 794790 900879 0.531282343M2 1261568 1780689 0.5853184 M3 1165156 1427840 0.550653 Mean (M1-M3)0.555751248 STDEV 0.027376412

The methods described herein used two distinct amplification reactionsto amplify the loci of interest. In the first PCR reaction,oligonucleotides were designed to anneal upstream and downstream of theloci of interest. Unlike traditional genomic amplification, theseprimers were not degenerate and annealed at a specified distance fromthe loci of interest. However, due to the length of the primers, it islikely that the primers annealed to other regions of the genome. Theseprimers were used to increase the amount of DNA available for geneticanalysis.

The second PCR reaction employs the methods described in Examples 1-6.The primers are designed to amplify the loci of interest, and thesequence is determined at the loci of interest. The conditions of thesecond PCR reaction allowed specific amplification of the loci ofinterest from the multiplexed template DNA. If there were anynon-specific products from the multiplex reaction, they did not impedeamplification of the loci of interest. There was no statisticaldifference in the percentages of allele 2 to allele 1 at the four SNPsanalyzed, regardless of whether the amplification was performed onoriginal template DNA or multiplexed template DNA.

The SNPs analyzed in this example were located on human chromosome 21.However, the methods can be applied to non-human and human DNA includingbut not limited to chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, and Y. The multiplex methodscan also be applied to analysis of genetic mutations including but notlimited to nucleotide substitutions, insertions, deletions, andrearrangements.

The above methods can be used to increase the amount of DNA availablefor genetic analysis whenever the starting template DNA is limiting inquantity. For example, pre-malignant and pre-invasive lesions withmalignant cells usually constitute a small fraction of the cells in thespecimen, which reduces the number of genetic analyses that can beperformed. The methods described herein can be used to increase theamounts of malignant DNA available for genetic analysis. Also, thenumber of fetal gnomes present in the maternal blood is often low; themethods described herein can be used to increase the amount of fetalDNA.

Example 13

Plasma isolated from blood of a pregnant female contains both maternaltemplate DNA and fetal template DNA. As discussed earlier, thepercentage of fetal. DNA in the maternal plasma varies for each pregnantfemale. However, the percentage of fetal DNA can be determined byanalyzing SNPs wherein the maternal template DNA is homozygous and thetemplate DNA obtained from the plasma displays a heterozygous pattern.

For example, assume SNP X can either be adenine or guanine, and thematernal DNA for SNP X is homozygous for guanine. The labeling methoddescribed in Example 6 can be used to determine the sequence of thetemplate DNA in the plasma sample. If the plasma sample contains fetalDNA, which is heterozygous at SNP X, the following DNA molecules areexpected after digestion with the type IIS restriction enzyme BsmF I,and the fill-in reaction with labeled ddGTP, unlabeled dATP, dTTP, anddCTP.

Maternal Allele 1 5′ GGGT G* 3′CCCA C T C A Maternal Allele 2 5′ GGGT G*3′CCCA C T C A Fetal Allele 1 5′ GGGT G* 3′CCCA C T C A Fetal Allele 25′ GGGT A A G* 3′CCCA T T C A

Two signals are seen; one signal corresponds to the DNA molecules filledin with ddGTP at position one complementary to the overhang and thesecond signal corresponds to the DNA molecules filled in with ddGTP atposition three complementary to the overhang. However, the maternal DNAis homozygous for guanine, which corresponds to the DNA molecules filledin at position one complementary to the overhang. The signal from theDNA molecules filled in with ddGTP at position three complementary tothe overhang corresponds to the adenine allele, which represents thefetal DNA. This signal becomes a beacon for the fetal DNA, and can usedto measure the amount of fetal DNA present in the plasma sample.

There is no difference in the amount of fetal DNA from one chromosome toanother. For instance, the percentage of fetal DNA in any givenindividual from chromosome 1 is the same as the percentage of fetal DNAfrom chromosome 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, X and Y. Thus, the allele ratio calculated for SNPson one chromosome can be compared to the allele ratio for the SNPs onanother chromosome.

For example, the allele ratio for the SNPs on chromosome 1 should beequal to the allele ratio for the SNPs on chromosomes 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, and Y.However, if the fetus has a chromosomal abnormality, including but notlimited to a trisomy or monosomy, the ratio for the chromosome that ispresent in an abnormal copy number will differ from the ratio for theother chromosomes.

Blood from a pregnant female was collected after informed consent hadbeen obtained. The blood sample was used to demonstrate that fetal DNAcan be detected in the maternal plasma by analyzing SNPs wherein thematernal DNA was homozygous, and the same SNP displayed a heterozygouspattern from DNA obtained from the plasma of a pregnant woman.

Preparation of Plasma from Whole Blood

Plasma was isolated from 4 tubes each containing 9 ml of blood (FischerScientific, 9 ml EDTA Vacuette tubes, catalog number NC9897284). Theblood was obtained by venipuncture from a pregnant female who had giveninformed consent. After collecting the blood, formaldehyde (25 μl/ml ofblood) was added to each of the tubes. The tubes were placed at 4° C.until shipment. The tubes were shipped via Federal Express in a foamcontainer containing an ice pack.

The blood was centrifuged at 1000 rpm for 10 minutes. The brake on thecentrifuge was not used. This centrifugation step was repeated. Thesupernatant was transferred to a new tube and spun at 3,000 rpm for tenminutes. The brake on the centrifuge was not used. The supernatant fromeach of the four tubes was pooled and aliquoted into two tubes. Theplasma was stored at −80° C. until the DNA was purified.

Template DNA was isolated using the QIAmp DNA Blood Midi Kit supplied byQIAGEN (Catalog number 51183). The template DNA was isolated as perinstructions included in the kit. The template DNA from the plasma waseluted in a final volume of 20 microliters.

Isolation of Maternal DNA

After the plasma was removed from the sample described above, onemilliliter of the remaining blood sample, which is commonly referred toas the “buffy-coat,” was transferred to a new tube. One milliliter of1×PBS was added to the sample. Template DNA was isolated using the QIAmpDNA Blood Midi Kit supplied by QIAGEN (Catalog number 51183).

Identification of Homozygous Maternal SNPs

Example 8 describes a method for identifying SNPs that are highlyvariable within the population or for identifying heterozygous SNPs fora given individual. The methods as described in Example 8 were appliedto the maternal template DNA to identify SNPs on chromosome 13 whereinthe maternal DNA was homozygous. Any number of SNPs can be screened. Thenumber of SNPs to be screened is proportional to the number ofheterozygous SNPs in the fetal DNA that need to be analyzed.

As described in detail in Example 6, one labeled nucleotide can be usedto determine the sequence of both alleles at a particular SNP. SNPs forwhich the sequence can be determined with labeled ddGTP in the presenceof unlabeled dATP, dTTP, and dCTP were chosen for this example. However,SNPs for which the sequence can be determined with labeled ddATP, ddCTPor ddTTP can also be used. Additionally, the SNPs to be analyzed can bechosen such that all are labeled with the same nucleotide or anycombination of the four nucleotides. For instance, if 400 SNPs are to bescreened, 100 can be chosen such that the sequence is determined withlabeled ddATP, 100 can be chosen such that the sequence is determinedwith labeled ddATP, 100 can be chosen such that the sequence isdetermined with labeled ddGTP, and 100 can be chosen such that thesequence is determined with labeled ddCTP, or any combination of thefour labeled nucleotides.

Twenty-nine SNPs wherein the maternal DNA was homozygous wereidentified: TSC0052277, TSC1225391, TSC0289078, TSC1349804, TSC0870209,TSC0194938, TSC0820373, TSC0902859, TSC0501510, TSC1228234, TSC0082910,TSC0838335, TSC0818982, TSC0469204, TSC1084457, TSC0466177, TSC1270598,TSC1002017, TSC1104200, TSC0501389, TSC0039960, TSC0418134, TSC0603688,TSC0129188, TSC1103570, TSC0813449, TSC0701940, TSC0087962, andTSC0660274. Heterozygous SNPs will vary from individual to individual.

Design of Multiplex Primers

A low copy number of fetal genomes typically is present in the maternalplasma. To increase the copy number of the loci of interest located onchromosome 13, primers were designed to anneal at approximately 130bases upstream and 130 bases downstream of each loci of interest. Thiswas done to reduce statistical sampling error that can occur whenworking with a low number of genomes, which can influence the ratio ofone allele to another (see Example 11). The primers were 12 bases inlength. However, primers of any length can be used including but, notlimited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36-45, 46-55, 56-65, 66-75, 76-85, 86-95, 96-105, 106-115, 116-125, andgreater than 125 bases. Primers were designed to anneal to both thesense strand and the antisense strand.

The primers were designed to terminate at the 3′ end in the dinucleotide“AA” to reduce the formation of primer-dimers. However, the primers canbe designed to end in any of the four nucleotides and in any combinationof the four nucleotides.

The multiplex primers for SNPTSC0052277 were

Forward primer: 5′ GACATGTTGGAA 3′ (SEQ ID NO: 513) Reverse primer:5′ ACTTCCAGTTAA 3′ (SEQ ID NO: 514)

The multiplex primers for SNP TSC1225391 were:

Forward primer: 5′ GTTTCCTGTTAA 3′ (SEQ ID NO: 515) Reverse primer5′ CGATGATGACAA 3′ (SEQ ID NO: 516)

The multiplex primers for SNP TSC0289078 were:

Forward primer 5′ GAGTAGAGACAA 3′ (SEQ ID NO: 517) Reverse primer5′ TCCCGGATACAA 3′ (SEQ ID NO: 518)

The multiplex primes for SNP TSC1349804 were:

Forward primer: 5′ CATCCTCTAGAA 3′ (SEQ ID NO: 519) Reverse primer:5′ TATTCCTGAGAA 3′ (SEQ ID NO: 520)

The multiplex primers for SNP TSC0870209 were:

Forward primer: 5′ AGTTTGTTTTAA 3′ (SEQ ID NO: 521) Reverse primer:5′ TATAAACGATAA 3′ (SEQ ID NO: 522)

The multiplex primers for SNP TSC0194938 were:

Forward primer: 5′ TTTGACCGATAA 3′ (SEQ ID NO: 523) Reverse primer:5′ TGACAGGACCAA 3′ (SEQ ID NO: 524)

The multiplex primers for SNP TSC0820373 were:

Forward primer: 5′ TTATTCATTCAA 3′ (SEQ ID NO: 525) Reverse primer:5′ AGTTTTTTCACAA 3′ (SEQ ID NO: 526)

The multiplex primers for SNP TSC0902859 were:

Forward primer: 5′ CACCTCCCTGAA 3′ (SEQ ID NO: 527) Reverse primer:5′ CCAGATTGAGAA 3′ (SEQ ID NO: 528)

The multiplex primers for SNP TSC0501510 were:

Forward primer: 5′ TGTGTCCACCAA 3′ (SEQ ID NO: 529) Reverse primer:5′ CTTCTATTCCAA 3′ (SEQ ID NO: 530)

The multiplex primers for SNP TSC1228234 were:

Forward primer: 5′ TCACAATAGGAA 3′ (SEQ ID NO: 531) Reverse primer5′ TACAAGTGAGAA 3′ (SEQ ID NO: 532)

The multiplex primers for SNP TSC0082910 were:

Forward primer: 5′ GAGTTTTCGTAA 3′ (SEQ ID NO: 533) Reverse primer:5′ GTGTGCCCCCAA 3′ (SEQ ID NO: 534)

The multiplex primers for SNP TSC0838335 were:

Forward primer: 5′ GCACCACTGCAA 3′ (SEQ ID NO: 535) Reverse primer:5′ GAACACAATGAA 3′ (SEQ ID NO: 536)

The multiplex primers for SNP TSC0818982 were:

Forward primer: 5′ TATCCTATTCAA 3′ (SEQ ID NO: 537) Reverse primer:5′ CAACCATTATAA 3′ (SEQ ID NO: 538)

The multiplex primers for SNP TSC0469204 were:

Forward primer: 5′ TATGCTTTACAA 3′ (SEQ ID NO: 539) Reverse primer:5′ TTTGTTTACCAA 3′ (SEQ ID NO: 540)

The multiplex primers for SNP TSC1084457 were:

Forward primer: 5′ AGGAAATTAGAA 3′ (SEQ ID NO: 541) Reverse primer:5′ TGTTAGACTTAA 3′ (SEQ ID NO: 542)

The multiplex primers for SNP TSC0466177 were:

Forward primer: 5′ TATTTGGAGGAA 3′ (SEQ ID NO: 543) Reverse primer:5′ GGCATTTGTCAA 3′ (SEQ ID NO: 544)

The multiplex primers for SNP TSC1270598 were:

Forward primer: 5′ ATACTCCAGGAA 3′ (SEQ ID NO: 545) Reverse primer:5′ CAGCCTGGACAA 3′ (SEQ ID NO: 546)

The multiplex primers for SNP TSC1002017 were:

Forward primer: 5′ CCATTGCAGTAA 3′ (SEQ ID NO: 547) Reverse primer:5′ AGGTTCTCATAA 3′ (SEQ ID NO: 548)

The multiplex primers for SNP TSC1104200 were:

Forward primer: 5′ TGTCATCATTAA 3′ (SEQ ID NO: 549) Reverse primer:5′ TGGTATTTGCAA 3′ (SEQ ID NO: 550)

The multiplex primers for SNP TSC0501389 were:

Forward primer: 5′ TAGGGTTTGTAA 3′ (SEQ ID NO: 551) Reverse primer:5′ CCCTAAGTAGAA 3′ (SEQ ID NO: 552)

The multiplex primers for SNP TSC0039960 were:

Forward primer: 5′ GTATTTCTTTAA 3′ (SEQ ID NO: 553) Reverse primer:5′ GAGTCTTCCCAA 3′ (SEQ ID NO: 554)

The multiplex primers for SNP TSC0418134 were:

Forward primer: 5′ CAGGTAGAGTAA 3′ (SEQ ID NO: 555) Reverse primer:5′ ATAGGATGTGAA 3′ (SEQ ID NO: 556)

The multiplex primers for SNP TSC0603688 were:

Forward primer: 5′ CAATGTGTATAA 3′ (SEQ ID NO: 557) Reverse primer:5′ AGAGGGCATCAA 3′ (SEQ ID NO: 558)

The multiplex primers for SNP TSC0129188 were:

Forward primer: 5′ CCAGTGGTCTAA 3′ (SEQ ID NO: 559) Reverse primer:5′ TAAACAATAGAA 3′ (SEQ ID NO: 560)

The multiplex primers for SNP TSC1103570 were:

Forward primer: 5′ GCACACTTTTAA 3′ (SEQ ID NO: 561) Reverse primer:5′ ATGGCTCTGCAA 3′ (SEQ ID NO: 562)

The multiplex primers for SNP TSC0813449 were:

Forward primer: 5′ GTCATCTTGTAA 3′ (SEQ ID NO: 563) Reverse primer:5′ TGCTTCATCTAA 3′ (SEQ ID NO: 564)

The multiplex primers for SNP TSC0701940 were:

Forward primer: 5′ AGAAAGGGGCAA 3′ (SEQ ID NO: 565) Reverse primer:5′ CTTTTCTTTCAA 3′ (SEQ ID NO: 566)

The multiplex primers for SNP TSC0087962 were:

Forward primer: 5′ CTACTCTCTCAA 3′ (SEQ ID NO: 567) Reverse primer:5′ ACAGCATTATAA 3′ (SEQ ID NO: 568)

The multiplex primers for SNP TSC0660274 were:

Forward primer: 5′ ACTGCTCTGGAA 3′ (SEQ ID NO: 569) Reverse primer:5′ GCAGAGGCACAA 3′ (SEQ ID NO: 570)

Multiplex PCR

Regions on chromosome 13 surrounding the above-mentioned 29 SNPs wereamplified from the template genomic DNA using the polymerase chainreaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporatedherein by reference). This PCR reaction used primers that annealedapproximately 150 bases upstream and downstream of each loci ofinterest. The fifty-eight primers were mixed together and used in asingle reaction to amplify the template DNA. This reaction was done toincrease the number of copies of the loci of interest, which eliminateserror generated from a low number of genomes.

For increased specificity, a “hot-start” PCR reaction was used. PCRreactions were performed using the HotStarTaq Master Mix Kit supplied byQIAGEN (catalog number 203443). The amount of template DNA and primerper reaction can be optimized for each locus of interest. In thisexample, the 20 μl of plasma template DNA was used.

Two microliters of each forward and reverse primer, at concentrations of5 mM were pooled into a single microcentrifuge tube and mixed. Fourmicroliters of the primer mix was used in a total PCR reaction volume of50 μl (20 μl of template plasma DNA, 1 μl of sterile water, 4 μl ofprimer mix, and 25 μl of HotStar Taq. Twenty-five cycles of PCR wereperformed. The following PCR conditions were used:

-   -   (1) 95° C. for 15 minutes;    -   (2) 95° C. for 30 second;    -   (3) 4° C. for 30 seconds;    -   (4) 37° C. for 30 seconds;    -   (5) Repeat steps 2-4 twenty-four (24) times;    -   (6) 72° C. for 10 minutes.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results.

Other methods of genomic amplification can also be used to increase thecopy number of the loci of interest including but not limited to primerextension preamplification (PEP) (Zhang et al., PNAS, 89:5847-51, 1992),degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius, et al.,Genomics 13:718-25, 1992), strand displacement amplification using DNApolymerase from bacteriophage 29, which undergoes rolling circlereplication (Dean et al., Genomic Research 11:1095-99, 2001), multipledisplacement amplification (U.S. Pat. No. 6,124,120), REPLI-g™ WholeGenome Amplification kits, and Tagged PCR.

Purification of Fragment of Interest

The unused primers, and nucleotides were removed from the reaction byusing Qiagen MinElute PCR purification kits (Qiagen, Catalog Number28004). The reactions were performed following the manufacturer'sinstructions supplied with the columns. The DNA was doted in 100 μl ofsterile water.

PCR Reaction Two Design of Primers

SNPTSC0052277 was amplified using the following primer set:

First primer: (SEQ ID NO: 571) 5′ CTCCGTGGTATGGAATTCCACTCAAATCTTCATTCAGA3′ Second primer: (SEQ ID NO: 572) 5′ ACGTCGGGTTACGGGACACCTGATTCCTC 3′

SNP TSC1225391 was amplified using the following primer set:

First primer: (SEQ ID NO: 573) 5′ TACCATTGGTTTGAATTCTTGTTTCCTGTTAACCATGC3′ Second primer: (SEQ ID NO: 574) 5′ GCCGAGTTCTACGGGACAGAAAAGGGAGC 3′

SNP TSC0289078 was amplified using the following primer set:

First primer: (SEQ ID NO: 575) 5′ TGCAGTGATTTCGAATTCGAGACAATGCTGCCCAGTCA3′ Second primer: (SEQ ID NO: 576) 5′ TCTAAATTCTCTGGGACCATTCCTTCAAC 3′

SNP TSC1349804 was amplified using the following primer set:

First primer: (SEQ ID NO: 577) 5′ ACTAACAGCACTGAATTCCATGCTCTTGGACTTTCCAT3′ Second primer: (SEQ ID NO: 578) 5′ TCCCCTAACGTTGGGACACAGAATACTAC 3′

SNP TSC0870209 was amplified using the following primer set:

First primer: (SEQ ID NO: 579) 5′ GTCGACGATGGCGAATTCCTGCCACTCATTCAGTTAGC3′ Second primer: (SEQ ID NO: 580) 5′ GAACGGCCCACAGGGACCTGGCATAACTC 3′

SNP TSC0194938 was amplified using the following primer set:

First primer: (SEQ ID NO: 581) 5′ TCATGGTAGCAGGAATTCTGCTTTGACCGATAAGGAGA3′ Second primer: (SEQ ID NO: 582) 5′ ACTGTGGGATTCGGGACTGTCTACTACCC 3′

SNP TSC0820373 was amplified using the following primer set:

First primer: (SEQ ID NO: 583) 5′ ACCTCTCGGCCGGAATTCGGAAAAGTGTACAGATCATT3′ Second primer: (SEQ ID NO: 584) 5′ GCCGGATACGAAGGGACGGCTCGTGACTC 3′

SNP TSC0902859 was amplified using the following primer set:

First primer: (SEQ ID NO: 585) 5′ CCGTAGACTAAAGAATTCCCTGATGTCAGGCTGTCACC3′ Second primer: (SEQ ID NO: 586) 5′ ATCGGATCAGTCGGGACGGTGTCTTTGCC 3′

SNP TSC0501510 was amplified using the following primer set:

First primer: (SEQ ID NO: 587) 5′ GCATAGGCGGGAGAATTCCCTGTGTCCACCAAAGTCGG3′ Second primer: (SEQ ID NO: 588) 5′ CCCACATAGGGCGGGACAAAGAGCTGAAC 3′

SNP TSC1228234 was amplified using the following primer set:

First primer: (SEQ ID NO: 589) 5′ GGCTTGCCGAGCGAATTCTAGGAAAGATACGGAATCAA3′ Second primer: (SEQ ID NO: 590) 5′ TAACCCTCATACGGGACTTTCATGGAAGC 3′

SNP TSC0082910 was amplified using the following primer set:

First primer: (SEQ ID NO: 591) 5′ ATGAGCACCCGGGAATTCTGATTGGAGTCTAGGCCAAA3′ Second primer: (SEQ ID NO: 592) 5′ TGCTCACCTTCTGGGACGTGGCTGGTCTC 3′

SNP TSC0838335 was amplified using the following primer set:

First primer: (SEQ ID NO: 593) 5′ ACCGTCTGCCACGAATTCTGGAAAACATGCAGTCTGGT3′ Second primer: (SEQ ID NO: 594) 5′ TACACGGGAGGCGGGACAGGGTGATTAAC 3′

SNP TSC0818982 was amplified using the following primer set:

First primer: (SEQ ID NO: 595) 5′ CTTAAAGCTAACGAATTCAGAGCTGTATGAAGATGCTT3′ Second primer: (SEQ ID NO: 596) 5′ AACGCTAAAGGGGGGACAACATAATTGGC 3′

SNP TSC0469204 was amplified using the following primer set:

First primer: (SEQ ID NO: 597) 5′ TTGTAAGAACGAGAATTCTGCAACCTGTCTTTATTGAA3′ Second primer: (SEQ ID NO: 598) 5′ CTTCACCACTTTGGGACACTGAAGCCAAC 3′

SNP TSC1084457 was amplified using the following primer set:

First primer: (SEQ ID NO: 599) 5′ AACCATTGATTTGAATTCGAAATGTCCACCAAAGTTCA3′ Second primer: (SEQ ID NO: 600) 5′ TGTCTAGTTCCAGGGACGCTGTTACTTAC 3′

SNP TSC0466177 was amplified using the following primer set:

First primer: (SEQ ID NO: 601) 5′ CGAAGGTAATGTGAATTCTGCCACAATTAAGACTTGGA3′ Second primer: (SEQ ID NO: 602) 5′ ATACCGGTTTTCGGGACAGATCCATTGAC 3′

SNP TSC1270598 was amplified using the following primer set:

First primer: (SEQ ID NO: 603) 5′ CCTGAAATCCACGAATTCCACCCTGGCCTCCCAGTGCA3′ Second primer: (SEQ ID NO: 604) 5′ TAGATGGTAGGTGGGACAGGACTGGCTTC 3′

SNP TSC1002017 was amplified using the following primer set:

First primer: (SEQ ID NO: 605) 5′ GCATATCTTAGCGAATTCCTGTGACTAATACAGAGTGC3′ Second primer: (SEQ ID NO: 606) 5′ CCAAATATGGTAGGGACGTGTGAACACTC 3′

SNP TSC1104200 was amplified using the following primer set:

First primer: (SEQ ID NO: 607) 5′ TGCCGCTACAGGGAATTCATATGGCAGATATTCCTGAA3′ Second primer: (SEQ ID NO: 608) 5′ ACGTTGCGGACCGGGACTTCCACAGAGCC 3′

SNP TSC0501389 was amplified using the following primer set:

First primer: (SEQ ID NO: 609) 5′ CTTCGCCCAATGGAATTCGGTACAGGGGTATGCCTTAT3′ Second primer: (SEQ ID NO: 610) 5′ TGCACTTCTGCCGGGACCAGAGGAGAAAC 3′

SNP TSC0039960 was amplified using the following primer set:

First primer: (SEQ ID NO: 611) 5′ TGTGGGTATTCTGAATTCCACAAAATGGACTAACACGC3′ Second primer: (SEQ ID NO: 612) 5′ ACGTCGTTCAGTGGGACATTAAAAGGCTC 3′

SNP TSC0418134 was amplified using the following primer set:

First primer: (SEQ ID NO: 613) 5′ GGTTATGTGTCAGAATTCTGAAACTAGTTTGGAAGTAC3′ Second primer: (SEQ ID NO: 614) 5′ GCCTCAGTTTCGGGGACAGTTCTGAGGAC 3′

SNP TSC0603688 was amplified using the following primer set:

First primer: (SEQ ID NO: 615) 5′ TGTAACACGGCCGAATTCCTCATTTGTATGAAATAGGT3′ Second primer: (SEQ ID NO: 616) 5′ AATCTAACTTGAGGGACCGGCACACACAC 3′

SNP TSC0129188 was amplified using the following primer set:

First primer: (SEQ ID NO: 617) 5′ AGTGTCCCCTTAGAATTCGCAGAGACACCACAGTGTGC3′ Second primer: (SEQ ID NO: 618) 5′ TTTGCTACAGTCGGGACCCTTGTGTGCTC 3′

SNP TSC1103570 was amplified using the following primer set:

First primer: (SEQ ID NO: 619) 5′ AGCACATCACTAGAATTCAATACCATGTGTGAGCTCAA3′ Second primer: (SEQ ID NO: 620) 5′ AATCCTGCTTCCGGGACCTAACTTTGAAC 3′

SNP TSC0813449 was amplified using the following primer set:

First primer: (SEQ ID NO: 621) 5′ TTTCATTTTCTGGAATTCCTCTAATGATTTTCTGGAGC3′ Second primer: (SEQ ID NO: 622) 5′ CGTCGCCGCGTAGGGACTTTTTCTTCCAC 3′

SNP TSC0701940 was amplified using the following primer set:

First primer: (SEQ ID NO: 623) 5′TTACTTAATCCTGAATTCGAGAAAAGCCATGTTGATAA 3′ Second primer:(SEQ ID NO: 624) 5′ TCATGGGTCGCTGGGACTTTGCCCTCTGC 3′

SNP TSC0087962 was amplified using the following primer set:

First primer: (SEQ ID NO: 625) 5′ACTAACAGCACTGAATTCATTTTACTATAATCTGCTAC 3′ Second primer:(SEQ ID NO: 626) 5′ GTTAGCCGAGAAGGGACTGTCTGTGAAGC 3′

SNP TSC0660274 was amplified using the following primer set:

First primer: (SEQ ID NO: 627) 5′AAATATGCAGCGGAATTCGTAAGTGACCTATTAATAAC 3′ Second primer:(SEQ ID NO: 628) 5′ GCGATGGTTACGGGGACAGCCAGGCAACC 3′

Each first primer had a biotin tag at the 5′ end and contained arestriction enzyme recognition site for EcoRI, and was designed toanneal at a specified distance from the locus of interest. This allows asingle reaction to be performed for the loci of interest, as each lociof interest will migrate at a distinct position (based on annealingposition of first primer). The second primer contained a restrictionenzyme recognition site for BsmF I.

All loci of interest were amplified from the multiplexed template DNAusing the polymerase chain reaction (PCR, U.S. Pat. Nos. 4,683,195 and4,683,202, incorporated herein by reference). In this example, the lociof interest were amplified in separate reaction tubes but they couldalso be amplified together in a single PCR reaction. For increasedspecificity, a “hot-start” PCR was used. PCR reactions were performedusing the HotStarTaq Master Mix Kit supplied by QIAGEN (catalog number203443).

The amount of multiplexed template DNA and primer per reaction can beoptimized for each locus of interest. One microliter of the multiplexedtemplate DNA eluted from the MinElute column was used in the PCRreaction for each locus of interest, and 5 μM of each primer was used.The twenty-nine SNPs described above also were amplified from thematernal DNA (15 ng of DNA was used in the PCR reaction; primerconcentrations were as stated above). Forty cycles of PCR wereperformed. The following PCR conditions were used:

-   -   (1) 95° C. for 15 minutes and 15 seconds;    -   (2) 37° C. for 30 seconds;    -   (3) 95° C. for 30 seconds;    -   (4) 57° C. for 30 seconds;    -   (5) 95° C. for 30 seconds;    -   (6) 64° C. for 30 seconds;    -   (7) 95° C. for 30 seconds;    -   (8) Repeat steps 6 and 7 thirty nine (39) times;    -   (9) 72° C. for 5 minutes.

In the first cycle of PCR, the annealing temperature was about themelting temperature of the 3′ annealing region of the second primers,which was 37° C. The annealing temperature in the second cycle of PCRwas about the melting temperature of the 3′ region, which anneals to thetemplate DNA, of the first primer, which was 57° C. The annealingtemperature in the third cycle of PCR was about the melting temperatureof the entire sequence of the second primer, which was 64° C. Theannealing temperature for the remaining cycles was 64° C. Escalating theannealing temperature from TM1 to TM2 to TM3 in the first three cyclesof PCR greatly improves specificity. These annealing temperatures arerepresentative, and the skilled artisan will understand the annealingtemperatures for each cycle are dependent on the specific primers used.

The temperatures and times for denaturing, annealing, and extension, canbe optimized by trying various settings and using the parameters thatyield the best results. In this example, the first primer was designedto anneal at various distances from the locus of interest. The skilledartisan understands that the annealing location of the first primer canbe 5-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55,56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101-105,106-110, 111-115, 116-120, 121-125, 126-130, 131-140, 140-160, 160-180,180-200, 200-220, 220-240, 240-260. 260-280. 280-300, 300-350, 350-400,400-450, 450-500, or greater than 500 bases from the locus of interest.

Purification of Fragment of Interest

The PCR products were separated from the genomic template DNA. Each PCRproduct was placed into a well of a Streptawell, transparent, High-Bindplate from Roche Diagnostics GmbH (catalog number 1 645 692, as listedin Roche Molecular Biochemicals, 2001 Biochemicals Catalog).Alternatively, the PCR products can be pooled into a single well becausethe first primer was designed to allow the loci of interest to separatebased on molecular weight. The first primers contained a 5′ biotin tagso the PCR products bound to the Streptavidin coated wells while thegenomic template DNA did not. The streptavidin binding reaction wasperformed using a Thermomixer (Eppendorf) at 1000 rpm for 20 min. at 37°C. Each well was aspirated to remove unbound material, and washed threetimes with 1×PBS, with gentle mixing (Kandpal et al., Nucl. Acids Res.18:1789-1795 (1990); Kaneoka et al., Biotechniques 10:30-34 (1991);Green et al., Nucl. Acids Res. 18:6163-6164 (1990)).

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme BsmFI, which binds to the recognition site incorporated into the PCRproducts from the second primer. The digests were performed in theStreptawells following the instructions supplied with the restrictionenzyme. After digestion, the wells were washed three times with PBS toremove the cleaved fragments.

Incorporation of Labeled Nucleotide

The restriction enzyme digest with BsmF I yielded a DNA fragment with a5′ overhang, which contained the SNP site or locus of interest and a 3′recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide or nucleotides in the presence of a DNApolymerase.

As demonstrated in Example 6, the sequence of both alleles of a SNP canbe determined by filling in the overhang with one labeled nucleotide inthe presence of the other unlabeled nucleotides. The followingcomponents were added to each fill in reaction: 1 μl of fluorescentlylabeled ddGTP, 0.5 μl of unlabeled ddNTPs (40 μM), which contained allnucleotides except guanine, 2 μl of 10× sequenase buffer, 0.25 μl ofSequenase, and water as needed for a 20 μl reaction. The fill inreaction was performed at 40° C. for 10 min. Non-fluorescently labeledddNTP was purchased from Fermentas Inc. (Hanover, Md.). All otherlabeling reagents were obtained from Amersham (Thermo Sequenase DyeTerminator Cycle Sequencing Core Kit, US 79565).

After labeling, each Streptawell was rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments were then released from theStreptawells by digestion with the restriction enzyme EcoRI, accordingto the manufacturer's instructions that were supplied with the enzyme.Digestion was performed for 1 hour at 37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

After release from the streptavidin matrix, the sample was loaded into alane of a 36 cm 5% acrylamide (urea) gel (BioWhittaker MolecularApplications, Long Ranger Run Gel Packs, catalog number 50691). Thesample was electrophoresed into the gel at 3000 volts for 3 min. The gelwas run for 3 hours on a sequencing apparatus (Hoefer SQ3 Sequencer).The gel was removed from the apparatus and scanned on the Typhoon 9400Variable Mode Imager. The incorporated labeled nucleotide was detectedby fluorescence.

Below a schematic of the 5′ overhang for SNP TSC0838335 is depicted. Theentire sequence is not reproduced, only a portion to depict the overhang(where R indicates the variable site).

10/14 5′ TAA 3′ ATT R A C A Overhang position 1 2 3 4

The observed nucleotides for TSC0838335 are adenine and guanine on the5′ sense strand (herein depicted as the top strand). The nucleotide inposition three of the overhang corresponded to cytosine, which iscomplementary to guanine. Labeled ddGTP can be used to determine thesequence of both allele in the presence of unlabeled dATP, dCTP, anddTTP.

The restriction enzyme BsmF I was used to create the 5′ overhang, whichtypically cuts 10/14 from the recognition site. At times, BsmF I willcut 11/15 from the recognition site and generate the following overhang:

11/15 5′ TA 3′ AT T R A C Overhang position 0 1 2 3

Position 0 in the overhang is thymidine, which is complementary toadenine. Position 0 complementary to the overhang was filled in withunlabeled dATP, and thus after the fill-in reaction, the exact samemolecules were generated whether the enzyme cut at 10/14 or 11/15 fromthe recognition site. The DNA molecules generated after the fill-inreaction are depicted below:

G allele 10/14 5′ TAA  G* 3′ ATT C A C A Overhang position 1 2 3 4G allele 11/15 5′ TA A  G* 3′ AT T C A C Overhang position 0 1 2 3A allele 10/14 5′ TAA A T  G* 3′ ATT T A C A Overhang position 1 2 3 4A allele 11/15 5′ TA A A T  G* 3′ AT T T A C Overhang position 0 1 2 3

The maternal template DNA amplified for TSC0838335 displayed a singleband that migrated at the expected position of the higher molecularweight band, which corresponded to the “A” allele (see FIG. 20, lane 1).The maternal template DNA was homozygous for adenine at SNP TSC0838335.

However, in lane 2, amplification of the multiplexed template DNA forTSC0838335 isolated from the plasma of the same individual displayed twobands; a lower molecular weight band, which corresponded to the “G”allele, and the higher molecular weight band, which corresponded to the“A” allele. The template DNA isolated from the plasma of a pregnantfemale contains both maternal template DNA and fetal template DNA.

As seen in FIG. 20, lane 1, the maternal template DNA was homozygous foradenine at this SNP (compare lanes 1 and 2). The “G” allele representedthe fetal DNA. Signals from the maternal template DNA and the fetaltemplate DNA clearly have been distinguished. The “G” allele becomes abeacon for the fetal DNA and can be used to measure the amount of fetalDNA present in the sample. Additionally, once the percentage of fetalDNA in the maternal plasma for a given sample has been determined, anydeviation from this percentage indicates a chromosomal abnormality. Thismethod provides the first non-invasive method for the detection of fetalchromosomal abnormalities.

As seen in FIG. 20, lane 3, analysis of the maternal DNA for SNPTSC0418134 generated a single band that migrated at the expectedposition of the higher molecular weight band, which corresponded to theadenine allele. Likewise, analysis of the multiplexed template DNAisolated from the maternal plasma gave a single band, which migrated atthe expected position of the adenine allele (see FIG. 20, lane 4). Boththe maternal DNA and the fetal DNA are homozygous for adenine atTSC0418134.

Below, a schematic of the 5′ overhang for TSC0129188 is depicted,wherein R indicates the variable site;

10/14 5′ TCAT 3′ AGTA R A C T Overhang position 1 2 3 4

The nucleotide upstream of the variable site (R) does not correspond toguanine on the sense strand. Thus, the 5′ overhang generated by the11/15 cutting properties of BsmF I will be filled-in identically to the5′ overhang generated by the 10/14 cut. Labeled ddGTP in the presence ofunlabeled dATP, dTTP, and dCTP was used for the fill-in reaction. TheDNA molecules generated after the fill-in reaction are depicted below:

A allele 10/14 5′ TCAT A T  G* 3′ AGTA T A C T Overhang position 1 2 3 4G allele 10/14 5′ TCAT  G* 3′ AGTA C A C T Overhang position 1 2 3 4

Analysis of the maternal DNA for SNP TSC0129188 gave a single band thatcorresponded to the DNA molecules filled in with ddGTP at position 1complementary to the overhang, which represented the “G” allele (seeFIG. 20, lane 5). No band was detected for adenine allele, indicatingthat the maternal DNA is homozygous for guanine.

In contrast, analysis of the multiplexed template DNA from the maternalplasma, which contains both maternal DNA, and fetal DNA, gave twodistinct bands (see FIG. 20, lane 6). The lower molecular weight bandcorresponded to the “G” allele, while the higher molecular weightcorresponded to the “A” allele. The “A” allele represents the fetal DNA.Thus, a method has been developed that allows separation of maternal DNAand fetal DNA signals without the added complexity of having to isolatefetal cells. In addition, a sample of paternal DNA is not required todetect differences between the maternal DNA and the fetal DNA.

Analysis of the maternal DNA for SNP TSC0501389 gave a single band thatmigrated at the higher molecular weight position, which corresponded tothe “A” allele. No band was detected that corresponded to the “G”allele. Similarly, analysis of the multiplexed template DNA from thematernal plasma for SNP TSC0501389 gave a single band that migrated atthe higher molecular weight position, which corresponded to the “A”allele. Both the maternal template DNA and the fetal template DNA werehomozygous for adenine at SNP TSC0501389.

The maternal DNA and the template DNA from the plasma originated fromthe same sample. One sample, which was obtained through a non-invasiveprocedure, provided a genetic fingerprint for both the mother and thefetus.

Of the twenty-nine SNPs for which the maternal template DNA washomozygous, the fetal template DNA was heterozygous at two of thetwenty-nine SNPs. The fetal DNA was homozygous for the same allele asthe maternal template DNA at the remaining 27 SNPs (data not shown).Comparing the homozygous allele of the maternal template DNA and theplasma template DNA at a given SNP provides an added level of qualitycontrol. It is not possible that the maternal template DNA and theplasma template DNA are homozygous for different alleles at the sameSNP. If this is seen, it would indicate that an error in processing hadoccurred.

The methods described herein demonstrate that the maternal geneticsignal can be separated and distinguished from the fetal genetic signalin a maternal plasma sample. The above-example analyzed SNPs located onchromosome 13, however any chromosome can be analyzed including humanchromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, X and Y and fetal chromosomes 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X and Y.

In addition, the methods described herein can be used to detect fetalDNA in any biological sample including but not limited to cell, tissue,blood, serum, plasma, saliva, urine, tears, vaginal secretions,umbilical cord blood, chorionic villi, amniotic fluid, embryonictissues, lymph fluid, cerebrospinal fluid, mucosa secretions, peritonealfluid, ascitic fluid, fecal matter, or body exudates.

The methods described herein demonstrate that the percentage of fetalDNA in the maternal sample can be determined by analyzing SNPs whereinthe maternal DNA is homozygous, and the DNA isolated from the plasma ofthe pregnant female is heterozygous. The percentage of fetal DNA can beused to determine if the fetal genotype has any chromosomal disorders.

For example, if the percentage of fetal DNA present in the sample iscalculated to be 30% by analysis of chromosome 1 (chromosomalabnormalities involving chromosome 1 terminate early in the pregnancy),then any deviation from 30% fetal DNA is indicative of a chromosomalabnormality. For example, if upon analysis of a SNP or multiple SNPs onchromosome 18, the percentage of fetal DNA is higher than 30%, thiswould indicate that an additional copy of chromosome 18 is present. Thecalculated percentage of fetal DNA from any chromosome can be comparedto any other chromosome. In particular, the percentage of fetal DNA onchromosome 13 can be compared to the percentage of fetal DNA onchromosomes 18 and 21.

This analysis is assisted by knowledge of the expected ratio of oneallele to the other allele at each SNP. As discussed in Example 9, notall heterozygous SNPs display ratios of 50:50. Knowledge of the expectedratio of one allele to the other reduces the overall number of variablesites that must be analyzed. However, even without knowledge of theexpected ratios for the various SNPs, the percentage of fetal DNA can becalculated by analyzing a large number of SNPs. When the sampling sizeof SNPs is large enough, the statistical variation arising from thevalues of the expected ratios will be eliminated.

In addition, heterozygous maternal SNPs also provide valuableinformation. The analysis is not limited to homozygous maternal SNPs.For example, if at a heterozygous SNP on maternal DNA, the ratio ofallele 1 to allele 2 is 1:1, then in the plasma template DNA the ratioshould remain 1:1 unless the fetal DNA carries a chromosomalabnormality.

The above methods can also be used to detect mutations in the fetal DNAincluding but not limited to point mutations, transitions,transversions, translocations, insertions, deletions, and duplications.As seen in FIG. 20, fetal DNA can readily be distinguished from maternalDNA. The above methods can be used to determine the sequence of anylocus of interest for any gene.

Example 14

Plasma isolated from blood of a pregnant female contains both maternaltemplate DNA and fetal template DNA. As discussed above, fetalchromosomal abnormalities can be determined by analyzing SNPs whereinthe maternal template DNA is homozygous and the template DNA obtainedfrom the plasma displays a heterozygous pattern.

For example, assume SNP X can either be adenine or guanine, and thematernal DNA for SNP X is homozygous for guanine. The labeling methoddescribed in Example 6 can be used to determine the sequence of the DNAin the plasma sample. If the plasma sample contains fetal DNA, which isheterozygous at SNP X, the following DNA molecules are expected afterdigestion with the type IIS restriction enzyme BsmF I, and the fill-inreaction with labeled ddGTP, unlabeled dATP, dTTP, and dCTP.

Maternal Allele 1 5′ GGGT  G* 3′CCCA C T C A Maternal Allele 2 5′ GGGT G* 3′CCCA C T C A Fetal Allele 1 5′ GGGT  G* 3′CCCA C T C AFetal Allele 2 5′ GGGT A A  G* 3′CCCA T T C A

Two signals are seen; one signal corresponds to the DNA molecules filledin with ddGTP at position one complementary to the overhang and thesecond signal corresponds to the DNA molecules filled in with ddGTP atposition three complementary to the overhang. However, the maternal DNAis homozygous for guanine, which corresponds to the DNA molecules filledin at position one complementary to the overhang. The signal from theDNA molecules filled in with ddGTP at position three complementary tothe overhang corresponds to the adenine allele, which represents thefetal DNA. This signal becomes a beacon for the fetal DNA, and can usedto measure the amount of fetal DNA present in the plasma sample.

There is no difference in the amount of fetal DNA from one chromosome toanother. For instance, the percentage of fetal DNA in any givenindividual from chromosome 1 is the same as the percentage of fetal DNAfrom chromosome 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, X and Y. Thus, the allele ratio calculated for SNPson one chromosome can be compared to the allele ratio for the SNPs onanother chromosome.

For example, the allele ratio for the SNPs on chromosome 1 should beequal to the allele ratio for the SNPs on chromosomes 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, and Y.However, if the fetus has a chromosomal abnormality, including but notlimited to a trisomy or monosomy, the ratio for the chromosome that ispresent in an abnormal copy number will differ from the ratio for theother chromosomes.

To recapitulate the in vivo scenario of blood from a pregnant female,maternal DNA was mixed with DNA isolated from her child, who previouslywas diagnosed with Trisomy 21, in various ratios to represent varyingpercentages of fetal DNA. For example, to replicate the in vivo scenarioof 50% fetal DNA in maternal blood, equal amounts of maternal DNA weremixed with DNA isolated from her child with Down's syndrome. Thematernal DNA was analyzed to identify homozygous SNPs, and these SNPsthen were analyzed using the mixture of 50% maternal DNA and 50% Down'ssyndrome DNA. The ratio of allele 1 to allele 2 at heterozygous SNPs onchromosome 13 was compared to the ratio of allele 1 to allele 2 atheterozygous SNPs on chromosome 21.

Four different samples were analyzed: a sample with 100% of the DNA froma child with Down syndrome; a sample with 75% DNA from the child withDown syndrome and 25% DNA from the child's mother; a sample with 50% DNAfrom the child with Down syndrome and 50% DNA from the child's mother;and a sample with 40% DNA from the child with Down syndrome and 60% DNAfrom the child's mother. The maternal DNA was analyzed to identifyhomozygous SNPs. The DNA isolated from the child with Down syndrome wasgenotyped to identify heterozygous SNPs. Then, the samples weregenotyped at SNPs where the maternal DNA was homozygous and the DNA fromthe child was heterozygous. For each sample, these SNPs were analyzedten times.

Collection of Blood Samples

An Internal Review Board approved study was designed to allow collectionof blood samples from children afflicted with Down's syndrome and theirparents. For this study, blood was collected from the mother, thefather, and the child with Down's syndrome. Informed consent to collectblood from the child with Down's syndrome was granted by the parents aswell as the child. Blood was collected into 9 ml EDTA Vacuette tubes(catalog number NC9897284). The tubes were stored at 4° C. until readyfor processing.

Isolation of Plasma and Maternal Cells

The blood was stored at 4° C. until processing. The tubes were spun at1000 rpm for ten minutes in a centrifuge with braking power set to zero.The tubes were spun a second time at 1000 rpm for ten minutes. Thesupernatant (the plasma) of each sample was transferred to a new tubeand spun at 3000 rpm for ten minutes with the brake set to zero. Thesupernatant was transferred to a new tube and stored at −80° C.Approximately two milliliters of the “buffy coat,” which containsmaternal cells, was placed into a separate tube and stored at −80° C.

Isolation of DNA

DNA was isolated from the plasma sample using the Qiagen Midi Kit forpurification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA waseluted in 100 μl of distilled water. The Qiagen Midi Kit also was usedto isolate DNA from the maternal cells contained in the “buffy coat.”Maternal DNA and the plasma DNA were isolated from the same tube ofblood.

Identification of Maternal Homozygous SNPs

The maternal DNA was genotyped to identify homozygous SNPs. Sevenhundred and sixty-eight SNPs on chromosome 13 and 768 SNPs on chromosome21 were genotyped using the methods described in Example 6. Any numberof SNPs can be analyzed, and the SNPs can be located on human chromosome1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, X and Y. Preferably, the SNPs that are genotyped have allelefrequencies of 50:50, 60:40, 70:30, 80:20, or 90:10. As described inExample 8, the allele frequency of any given SNP can be determined.

Details regarding the SNPs located on chromosome 13 and 21 can be foundat the SNP consortium database, which can be accessed via the internetat http://www.snp.cshl.org. The primers were designed following theprocedures set fourth in the Examples described above, for example, inExamples 1, 2, 3, 5, and 6.

The first primers were designed so that after digestion with a Type IIsenzyme, the products had different molecular weights as described inExample 6. This allowed the amplified products to be pooled, and run ina single lane of a gel.

For example, the first primer can be designed such that after digestiona 30 base pair product is generated. Likewise, the first primer of adifferent locus of interest can be designed such that after digestion a40 base pair product is generated. The first primers can be designed sothat in a single reaction, numerous loci can be analyzed in one lane ofa gel (30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 base pair productscan be run in a single lane). The first primer can be designed to annealany distance from the locus of interest including but not limited tobetween 5-10, 10-25, 25-50, 50-75, 75-100, 100-150, 150-200, 200-250,250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650,650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000 andgreater than 1000 bases.

Amplification of the Loci of Interest

For each SNP that was genotyped, a PCR reaction was used to amplify theloci of interest. The PCR reactions were performed in 96-well plates.The first and second primer (3 μl of 1.25 μM stock concentration) foreach SNP was distributed into a well of a microtiter plate. Eight96-well PCR plates were set-up for chromosome 21 and eight 96-wellplates were set-up for chromosome 13. After the primers had beendistributed into the wells of the microtiter plates, a mixturecontaining the genomic DNA and HotStar PCR reagents was added to eachwell. Each PCR reaction contained 3 μl of each primer, 7.5 μl of HotStarTag Master mix, 0.5 μl of water, and 1 μl of genomic DNA (10 ng/μl).

The PCR cycling conditions were as follows:

(1) 95° C. for 15 minutes and 15 seconds;

(2) 37° C. for 30 seconds;

(3) 95° C. for 30 seconds;

(4) 52° C. for 30 seconds;

(5) 95° C. for 30 seconds;

(6) 58° C. for 30 seconds;

(7) 95° C. for 30 seconds;

(8) Repeat steps 6 and 7 thirty seven (37) times;

(9) 72° C. for 5 minutes.

Purification of Fragment of Interest

After the PCR reaction, 3 μl of a PCR product generated with a firstprimer designed to produce a 30 base pair product, 3 μl of a PCR productgenerated with a first primer designed to produce a 40 base pairproduct, 3 μl of a PCR product generated with a first primer designed toproduce a 50 base pair product, 3 μl of a PCR product generated with afirst primer designed to produce a 60 base pair product, 3 μl of a PCRproduct generated with a first primer designed to produce a 70 base pairproduct, 3 μl of a PCR product generated with a first primer designed toproduce a 80 base pair product, 3 μl of a PCR product generated with afirst primer designed to produce a 90 base pair product, 3 μl of a PCRproduct generated with a first primer designed to produce a 100 basepair product were mixed together in a well of a Streptawell,transparent, High-Bind plate from Roche Diagnostics GmbH (catalog number1 645 692, as listed in Roche Molecular Biochemicals, 2001 BiochemicalsCatalog). The first primers contained a 5′ biotin tag so the PCRproducts bound to the Streptavidin coated wells while the genomictemplate DNA did not. The streptavidin binding reaction was performedusing a Thermomixer (Eppendorf) at 1000 rpm for 20 min. at 37° C. Eachwell was aspirated to remove unbound material, and washed three timeswith 1×PBS, with gentle mixing (Kandpal et al., Nucl. Acids Res.18:1789-1795 (1990); Kaneoka et al., Biotechniques 10:30-34 (1991);Green et al., Nucl. Acids Res. 18:6163-6164 (1990)).

Restriction Enzyme Digestion of Isolated Fragments

The purified PCR products were digested with the restriction enzyme BsmFI, which binds to the recognition site incorporated into the PCRproducts from the second primer. The digests were performed in theStreptawells following the instructions supplied with the restrictionenzyme. After digestion, the wells were washed three times with PBS toremove the cleaved fragments.

Incorporation of Labeled Nucleotide

The restriction enzyme digest with BsmF I yielded a DNA fragment with a5′ overhang, which contained the SNP site or locus of interest and a 3′recessed end. The 5′ overhang functioned as a template allowingincorporation of a nucleotide or nucleotides in the presence of a DNApolymerase. As discussed in detail in Example 6, a single nucleotidelabeled with one chemical moiety can be used to determine the sequenceat a SNP.

The amplified loci of interest were pooled into the streptavidin-wellbased on size, and on the nucleotide used in the fill-in reaction. Thesequence of SNPs that were determined by using a guanine nucleotide werepooled together. Likewise, the sequence of SNPs that were determined byusing an adenine nucleotide were pooled together; the sequence of SNPsthat were determined by using a thymidine nucleotide were pooledtogether; and the sequence of SNPs that were determined by using acytosine nucleotide were pooled together.

Thus, a typical fill-in reaction contained 8 amplified loci, ranging insize of 30-120 base pair products; the sequence of all eight wasdetermined using a single nucleotide labeled with one chemical moiety.Any number of amplified loci can be pooled together.

The following components were added to each fill in reaction: 1 μl offluorescently labeled dideoxynucleotide (ddGTP for G fill-in reactions;ddATP for A fill-in reactions; ddTTP for thymidine fill-in reactions;and ddCTP for cytosine fill-in reactions), 0.5 μl of unlabeled dNTPs (40μM), which contained all nucleotides except the labeled nucleotide, 2 μlof 10× sequenase buffer, 0.25 μl of Sequenase, and water as needed for a20 μl reaction.

The fill in reaction was performed at 40° C. for 10 min.Non-fluorescently labeled dNTP was purchased from Fermentas Inc.(Hanover, Md.). All other labeling reagents were obtained from Amersham(Thermo Sequenase Dye Terminator Cycle Sequencing Core Kit, US 79565).

After labeling, each Streptawell was rinsed with 1×PBS (100 μl) threetimes. The “filled in” DNA fragments were then released from theStreptawells by digestion with the restriction enzyme EcoRI, accordingto the manufacturer's instructions that were supplied with the enzyme.Digestion was performed for 1 hour at 37° C. with shaking at 120 rpm.

Detection of the Locus of Interest

After release from the streptavidin matrix, the sample was loaded into alane of a 36 cm 5% acrylamide (urea) gel (BioWhittaker MolecularApplications, Long Ranger Run Gel Packs, catalog number 50691). Thesample was electrophoresed into the gel at 3000 volts for 3 min. The gelwas run for 3 hours on a sequencing apparatus (Hoefer SQ3 Sequencer).The gel was removed from the apparatus and scanned on the Typhoon 9400Variable Mode Imager. The incorporated labeled nucleotide was detectedby fluorescence. The homozygous SNPs were identified.

Identification of Heterozygous SNPs with the Trisomy 21 Template

The DNA isolated from the individual with Down syndrome (the child ofthe mother who was genotyped above) was analyzed to identifyheterozygous SNPs. The same seven hundred and sixty-eight SNPs onchromosome 13 and the same 768 SNPs on chromosome 21 that were analyzedwith the maternal DNA were genotyped using the methods described inExample 6. Any number of SNPs can be analyzed, and the SNPs can belocated on human chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, X or Y. Preferably, the SNPs thatare genotyped have allele frequencies of 50:50, 60:40, 70:30, 80:20, or90:10. As described in Example 8, the allele frequency of any given SNPcan be determined.

The process for genotyping the SNPS with the DNA isolated from theindividual with Down syndrome was as described for the maternal DNA. Theheterozygous SNPs were identified.

SNPs that were homozygous for the maternal DNA and heterozygous for theDNA isolated from the individual with Down syndrome were furtheranalyzed using samples that contained mixtures of maternal DNA and Downsyndrome DNA.

Generation of Samples Containing Maternal DNA and Down Syndrome DNA

The DNA and the DNA obtained from her child, who has Down's syndrome,were quantitated using a spectrophotometer. The maternal DNA and thechild's DNA were mixed together at various percentages to represent thesituation of circulating fetal DNA in the maternal blood. The followingpercentages were analyzed: 100% Down's syndrome DNA, 75% Down's syndromeDNA, 50% Down's syndrome DNA, and 40% Down's syndrome DNA.

The ratio at each heterozygous SNP was calculated by dividing the valueobtained for allele 1 by the value obtained for allele 2. For example,if SNP X can either be adenine (A) or guanine (G), the ratio at SNP Xwas calculated by dividing the value obtained for adenine by the valueobtained for guanine.

For the sample containing 100% Down syndrome DNA, sixty-two SNPs onchromosome 13, which were homozygous with the maternal DNA andheterozygous with the DNA isolated from the individual with Downsyndrome, were analyzed. For chromosome 21, forty-nine SNPs wereanalyzed that were homozygous with the maternal DNA and heterozygouswith the DNA isolated from the individual with Down syndrome.

The 62 SNPs on chromosome 13 and 49 SNPs on chromosome 21 were analyzedten separate times. As shown in Table XX, for each of the ten trials,the ratio of allele 1 to allele 2 on chromosome 13 was approximately 1.0as expected. For chromosome 13, there is one copy of allele 1 and onecopy of allele 2. The average of the ten trials was 1.051 with astandard deviation of 0.085.

With a Trisomy 21, there are two copies of one allele, which are usuallyinherited from the mother, and one copy of the other allele. Theexpected ratio is approximately 0.5 (one copy of allele 1/two copies ofallele 2). As shown in Table XX, the ratio for chromosome 21 varied froma low of 0.462 to a high of 0.634. For every trial, the ratio obtainedfor chromosome 21 was significantly distinct from the ratio obtained atchromosome 13. The average ratio for the ten trials was 0.531 with astandard deviation of 0.049.

The experiment was repeated ten times so that a true statisticalmeasurement could be obtained. If ten different genetic samples wereused, the SNPs that fit the criteria (maternal homozygous, Down syndromechild heterozygous) would be different, making it difficult to comparefrom sample to sample.

Statistical analysis revealed a confidence value of 99.9% that theratios obtained on chromosome 13 and on chromosome 21 represented truedifferences, rather than random numerical fluctuations in value. TheRavgen method identified the presence of the chromosomal abnormality.

For the sample containing 75% Down syndrome DNA and 25% maternal DNA,sixty two SNPS on chromosome 13 and fifty SNPs on chromosome 21 wereanalyzed, unless stated otherwise. For various trials, not all the SNPScould be quantitated because the bands corresponding to certain SNPswere faint. This may have been caused by poor PCR amplification, poorbinding to the streptavidin plate, or a weak fill-in reaction.

For trial 3, 61 SNPs on chromosome 13 were analyzed. For trail 4, 49SNPs were analyzed on chromosome 21. With regard to trial 5, 47 SNPs onchromosome 21 were analyzed and 61 SNPs on chromosome 13. For trial 7,49 SNPs were analyzed on chromosome 21 and 61 SNPs on chromosome 13. Fortrial 8, 49 chromosomes were analyzed on chromosome 21, and 59 SNPs wereanalyzed on chromosome 13. For trials 9 and 10, 59 SNPs on chromosome 13were analyzed.

The expected ratio on chromosome 13 for a heterozygous SNP is 0.6. Ifthe maternal chromosomes both contain an adenine nucleotide, and theDown syndrome genome is comprised of one chromosome with an adeninenucleotide and one chromosome with a guanine nucleotide, then the ratioof G:A is 0.75/(0.75 (Down syndrome A allele)+0.25+0.25 (maternal Aalleles)), which is 0.6. For the ten trials, the ratios obtained forchromosome 13 varied from 0.567 to 0.645. The average for the ten trialswas 0.609 with a standard deviation of 0.032 (see Table XX).

The expected ratio for chromosome 21 in a Trisomy condition is 0.375. Ifthe maternal chromosomes both contain an adenine nucleotide, and theDown syndrome genome is comprised of two chromosomes with an adeninenucleotide and one chromosome with a guanine nucleotide, then the ratioof G:A is 0.75/(0.75+0.75 (Down syndrome A alleles)+0.25+0.25 (maternalA alleles)), which is 0.375.

For the ten trials, the ratios obtained for chromosome 21 varied from0.350 to 0.4125, with an average of 0.384 and a standard deviation of0.017 (see Table XX). Statistical analysis revealed a confidence valueof 99.9% that the ratios obtained on chromosome 13 and on chromosome 21represented true differences, rather than random numerical fluctuationsin value. The Ravgen method identified the presence of the chromosomalabnormality in the presence of 25% maternal DNA.

With regard to the sample containing 50% Down syndrome DNA, 46 SNPs onchromosome 13 and 35 SNPs on chromosome 21 were analyzed, unless statedotherwise. For trial 1, 45 SNPs on chromosome 13 were analyzed. Fortrail 2, 44 SNPs on chromosome 13 were analyzed. For trial 3, 42 SNPs onchromosome 13 were analyzed. For trial 4, 44 SNPs on chromosome 13 and34 SNPs on chromosome 21 were analyzed. For trial 5, 34 SNPs onchromosome 21 were analyzed. For trials 7 and 8, 44 and 41 SNPs onchromosome 13, respectively, were analyzed. For trial 9, 44 SNPs onchromosome 13 and 34 SNPs on chromosome 21 were analyzed. For trial 10,44 SNPs on chromosome 13 were analyzed.

The expected ratio at a heterozygous SNP on chromosome 13 for the 50%sample is 0.33. If the maternal chromosomes both contain an adeninenucleotide, and the Down syndrome genome is comprised of one chromosomewith an adenine nucleotide and one chromosome with a guanine nucleotide,then the ratio of G:A is 0.50/(0.50 (Down syndrome A allele)+0.50+0.50(maternal A alleles)), which is 0.33. For the ten trials, the ratiosobtained for chromosome 13 varied from 0.302 to 0.347. The average forthe ten trials was 0.324 with a standard deviation of 0.0.13 (see TableXX).

The expected ratio for chromosome 21 in a Trisomy condition is 0.25. Ifthe maternal chromosomes both contain an adenine nucleotide, and theDown syndrome genome is comprised of two chromosomes with an adeninenucleotide and one chromosome with a guanine nucleotide, then the ratioof G:A is 0.50/(0.50+0.50 (Down syndrome A alleles)+0.50+0.50 (maternalA alleles)), which is 0.25.

For the ten trials, the ratios obtained for chromosome 21 varied from0.230 to 0.275, with an average of 0.244 and a standard deviation of0.015 (see Table XX). Statistical analysis revealed a confidence valueof 99.1% that the ratios obtained on chromosome 13 and on chromosome 21represented true differences, rather than random numerical fluctuationsin value. The Ravgen method identified the presence of the chromosomalabnormality in the presence of 50% maternal DNA.

For the sample containing 40% Down syndrome DNA, 60 SNPs on chromosome13 and 48 SNPs on chromosome 21 were analyzed, unless stated otherwise.For trial 1, 47 SNPs on chromosome 21 were analyzed. For trials 2-4, 59SNPs on chromosome 13 and 47 SNPs on chromosome 21 were analyzed. Fortrials 5 and 6, 46 SNPs on chromosome 21 were analyzed. For trail 7, 58SNPs on chromosome 13 were analyzed. For trial 8, 46 SNPs on chromosome21 were analyzed and for trials 9 and 10, 47 SNPs on chromosome 21 wereanalyzed.

The expected ratio at a heterozygous SNP on chromosome 13 for the 40%Down syndrome DNA sample is 0.25. If the maternal chromosomes bothcontain an adenine nucleotide, and the Down syndrome genome is comprisedof one chromosome with an adenine nucleotide and one chromosome with aguanine nucleotide, then the ratio of G:A is 0.40/(0.40 (Down syndrome Aallele)+0.60+0.60 (maternal A alleles)), which is 0.25. For the tentrials, the ratios obtained for chromosome 13 varied from 0.254 to0.285. The average for the ten trials was 0.269 with a standarddeviation of 0.009 (See Table XX).

The expected ratio for chromosome 21 in a Trisomy condition is 0.20. Ifthe maternal chromosomes both contain an adenine nucleotide, and theDown syndrome genome is comprised of two chromosomes with an adeninenucleotide and one chromosome with a guanine nucleotide, then the ratioof G:A is 0.40/(0.40+0.40 (Down syndrome A alleles)+0.60+0.60 (maternalA alleles)), which is 0.20.

For the ten trials, the ratios obtained for chromosome 21 varied from0.216 to 0.249, with an average of 0.23 and a standard deviation of0.011 (see Table XX). Statistical analysis revealed a confidence valueof 94.3% that the ratios obtained on chromosome 13 and on chromosome 21represented true differences, rather than random numerical fluctuationsin value. The Ravgen method identified the presence of the chromosomalabnormality in the presence of 60% maternal DNA.

The presence of the Trisomy 21 condition was identified with the Ravgenmethod in numerous samples that contained various percentages ofabnormal DNA. Each percentage of abnormal DNA was analyzed ten separatetimes and each time, the presence of the abnormal condition wasidentified. The ratio of allele 1 to allele 2 at multiple heterozygousSNPs on chromosome 13 was calculated, and the ratios were averaged. Thesame was done with the SNPs located on chromosome 21. The ratio obtainedfor the heterozygous SNPs on chromosome 13 was statistically differentfrom the ratio obtained on chromosome 21. The ratios obtained on bothchromosome 13 and 21 were near the mathematically predicted values.

In this example, the confidence interval for the samples with 100% Downsyndrome DNA and 75% Down syndrome DNA was 99.9%, and the confidenceinterval for the sample with 50% Down syndrome DNA was 99.1%, which isabout the accuracy reported for amniocentesis. The confidence intervalfor the sample containing 40% Down syndrome DNA was 94.3%, which is moreaccurate than currently marketed non-invasive tests for prenataldiagnostics.

As discussed above, about 60 SNPs on chromosome 13 and 50 SNPs onchromosome 21 were analyzed. To increase the confidence interval forsamples containing 40% fetal DNA or lower, a larger number of SNPs canbe analyzed. The Ravgen method provides a highly accurate,cost-effective way to sequence DNA, so sequencing a larger number ofSNPs is not difficult. The accuracy of the test is determined by thenumber of SNPs that are sequenced. For higher accuracy with samples thatcontain lower percentages of DNA, more SNPs can be analyzed.Alternatively, the methods described in this application can be used toensure that the samples contain a higher percentage of fetal DNA.

In this example, a sample containing 40% Down syndrome DNA, whichrepresented the fetal DNA in the maternal blood, was analyzed. Maternalblood samples with any percentage of fetal DNA can be analyzed includingbut not limited to 0.0001-1%, 1-10%, 10-20%, 20-30%, 30-40%, 40-50%,50-60%, 60-70%, 70-80%, 80-90%, and 90-100%.

TABLE XX The Ravgen method identifies chromosomal abnormalities inSamples containing 40% Down syndrome DNA Expected Expected Ratio atChrom. 13 21 21 13 21 21 100% DS DNA ~75% DS DNA Trial 1 0.959 0.57080.49 0.637 .3764 0.389 Trial 2 0.916 0.5024 0.48 0.567 .3894 0.362 Trial3 1029 0.4616 0.51 0.651 .3707 0.394 Trial 4 0.967 0.5123 0.491 0.580.3901 0.367 Trial 5 1.037 0.6339 0.51 0.645 .4125 0.392 Trial 6 1.1110.5425 0.53 0.645 .3743 0.392 Trial 7 1.154 0.495 0.54 0.594 .3974 0.373Trial 8 1.135 0.5276 0.532 0.583 .3901 0.368 Trial 9 1.148 0.5619 0.5340.579 .3899 0.367 Trial 10 1.057 0.4976 0.52 0.609 .350 0.378 AVG. 1.051.531 0.512 .609 0.384 0.378 STDEV .085 .049 .032 .017 ~50% DS DNA ~40%DS DNA Trial 1 0.347 0.275 0.258 0.277 0.239 0.217 Trial 2 0.3.16 0.2370.24 0.265 0.249 0.21 Trial 3 0.338 0.247 0.253 0.266 0.227 0.21 Trial 40.331 0.264 0.249 0.254 0.216 0.202 Trial 5 0.330 0.241 0.248 0.2740.246 0.215 Trial 6 0.324 0.240 0.244 0.268 0.22 0.211 Trial 7 0.3180.233 0.241 0.275 0.227 0.216 Trial 8 0.302 0.230 0.231 0.258 0.228 0.21Trial 9 0.315 0.238 0.240 0.285 0.231 0.222 Trial 10 0.318 0.235 0.2410.266 0.218 0.21 AVG. 0.324 0.244 0.244 0.269 0.23 0.212 STDEV 0.0130.015 0.009 0.011

Example 15

As discussed in Example 4 above, the use of cell lysis inhibitors, cellmembrane stabilizers, or cross-linking reagents can be used to increasethe percentage of fetal DNA in the maternal blood. In this example,methods for the isolation of free fetal DNA are disclosed, whichminimize the amount of maternal cell lysis. The effect of formalin onsixty-nine (69) maternal blood samples from twenty-seven clinicalpractices located in sixteen different states was analyzed. Formalin wasadded to all samples collected from the pregnant women, and thepercentage of fetal DNA was calculated using serial dilution analysisfollowed by PCR. A genetic marker on the Y chromosome was used tocalculate the percent of fetal DNA.

Collection of Blood Samples

In accordance with an IRS approved study, blood samples were collectedfrom pregnant women after informed consent had been granted. Bloodsamples were received from 27 different clinical sites operating in 16different states located throughout the U.S. Blood samples werecollected from both women carrying male and female fetuses, however,here, we report results obtained from woman carrying male fetuses, asthe Y chromosome is the accepted marker when quantitating percentages offetal DNA.

Blood is collected by any method or process that results in asubstantial increase in the ratio of fetal DNA/maternal DNA in theresulting serum or plasma after appropriate processing. As used herein,a substantial increase in the ratio of fetal DNA/maternal DNA is thatwhich can be detected by the methods as described herein. Such methodsor processes typically result in a substantial increase in the ratio offetal DNA/maternal DNA of about 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%,100% or more of the ratio of fetal DNA/maternal DNA found in bloodsamples collected by standard procedures.

In other embodiments, blood is collected by any method or process thatresults in a substantial increase in the amount of free fetal DNAcompared to the amount of total DNA recovered or detected in theresulting serum or plasma after processing. Such methods or processestypically result in a substantial increase so the fetal DNA recovered ordetected is about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more of the totalDNA recovered or detected in the processed plasma or serum sample.

All clinical sites were provided with a kit used for the venipunctureprocedure, which included 21 gauge needles, 9 ml EDTA Vacuette tubes(catalog number NC9897284) a syringe containing 0.225 ml of 10% neutralbuffered solution containing formaldehyde (4% w/v), an icepack, and ashipping container. The clinical sites were instructed to add theformaldehyde immediately after drawing the blood and to gently invertthe tubes.

The methods or processes of collecting blood samples may also includeother steps that result in lessened or reduced cell lysis. For instance,blood collection devices may be modified to decrease cell lysis due tosheer forces in the collection needle, syringe or tubes used. Forinstance, needles of large gauge may be employed to reduce cell sheeringor vacutainer tubes may be modified to reduce the velocity of bloodflow.

Isolation of Plasma

Any method may be used to isolate plasma from the cell components ofblood after collection but methods wherein cell lysis is substantiallyprevented, reduced or inhibited are preferred. The blood was stored at4° C. until processing. Methods for isolation of the plasma wereimplemented to reduce the amount of maternal cell lysis. The tubes werespun at 1000 rpm for ten minutes in a centrifuge with braking power andacceleration power set at zero to substantially prevent, reduce orinhibit cell lysis and or mixing of blood cell components into theplasma. The tubes were spun a second time at 1000 rpm for ten minuteswith braking power (centrifuge stopped by natural deceleration) andacceleration power set to zero. The supernatant (the plasma) of eachsample was transferred carefully to a new tube and spun at 3000 rpm forten minutes with the brake and acceleration power set at zero. Thesupernatant (the plasma) of each sample was collected via procedures tosubstantially prevent mixing of cell components into the plasma. Greatcare was taken to ensure that the buffy-coat was not disturbed. Apercentage of the supernatant can be left in the tube including but notlimited to 0.001-1%, 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%,60-70%, 70-80% or greater than 80%. In this example, about 0.5 ml of thesupernatant was left in the tube to ensure that the buffy-coat was notdisturbed. The supernatant was transferred to a new tube and stored at−80° C.

Isolation of DNA

DNA was isolated from the plasma sample using the Qiagen Midi Kit forpurification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183), DNA waseluted in 100 μl of distilled water. However, any method of DNAisolation can be used including cesium chloride gradients, gradients,sucrose gradients, glucose gradients, centrifugation protocols, boiling,Qiagen purification systems, QIA DNA blood purification kit, HiSpeedPlasmid Maxi Kit, QIAfilter plasmid kit, Promega DNA purificationsystems, MangeSil Paramagnetic Particle based, systems, Wizard SVtechnology, Wizard Genomic DNA purification kit, Amersham purificationsystems, GFX Genomic Blood DNA purification kit, Invitrogen LifeTechnologies Purification Systems, CONCERT purification system, Mo BioLaboratories purification systems, UltraClean BloodSpin Kits, andUlraClean Blood DNA Kit. The skilled artisan understands that themanufacturer's protocols can modified to increase the yield of DNA. Forexample, the Qiagen Midi Kit for purification of DNA recommends the useof 1×AL buffer. However, any concentration of AL buffer may be used ifthe yield of DNA increases including but not limited to 0.1-0.5×ALbuffer, 0.5-1×AL buffer, 1×-2×AL buffer, 2-3×AL buffer, 3-4×AL buffer,4-5×AL buffer, and greater than 5×AL buffer. The skilled artisanunderstands that the modifications and manipulations of the reagents arenot limited to AL buffer.

Quantification of Percentage of Fetal DNA

The percentage of fetal DNA present in the maternal plasma sample wascalculated using serial dilution analysis followed by PCR. Two differentsets of primers were used: one primer set was specific for the Ychromosome, and thus specific for fetal DNA, and the other primer setwas designed to amplify the cystic fibrosis gene, which is present onboth maternal template DNA and fetal template DNA.

Primer Design:

The following primers were designed to amplify the SRY gene on the Ychromosome:

Upstream primer: 5′ TGGCGATTAAGTCAAATTCGC 3′ (SEQ ID NO: 263)Downstream primer: 5′ CCCCCTAGTACCCTGACAATGTATT 3′ (SEQ ID NO: 264)

The following primers were designed to amplify the cystic fibrosis gene:

Upstream primer: 5′ CTGTTCTGTGATATTATGTGTGGT 3′ (SEQ ID NO: 265)Downstream primer: 5′ AATTGTTGGCATTCCAGCATTG 3′ (SEQ ID NO: 266)

PCR Reaction

The SRY gene and the cystic fibrosis gene were amplified from thetemplate genomic DNA using PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202).For increased specificity, a “hot-start” PCR was used. PCR reactionswere performed using the HotStarTaq Master Mix Kit supplied by Qiagen(Catalog No. 203443). For amplification of the SRY gene, the DNA elutedfrom the Qiagen purification column was diluted serially 1:2. Foramplification of the cystic fibrosis gene, the DNA eluted from theQiagen purification column was diluted 1:4, and then serially diluted1:2. The following components were used for each PCR reaction: 8 μl oftemplate DNA (diluted or undiluted), 1 μl of each primer (5 μM), 10 μlof HotStar Taq mix. The following PCR conditions were used:

(1) 95° C. for 15′

(2) 94° C. for 1′

(3) 54° C. for 15″

(4) 72° C. for 30″

(5) Repeat steps 2-4 for 45 cycles.

(6) 10′ at 72° C.

Amplification of the SRY gene was performed using the followingtemplates: undiluted, diluted 1:2, diluted 1:4, diluted 1:8, diluted1:16, diluted 1:32, diluted 1:64, diluted 1:128, diluted 1:256, anddiluted 1:512. Amplification of the cystic fibrosis gene was performedusing the following templates: diluted 1:4, diluted 1:8, diluted 1:16,diluted 1:32, diluted 1:64, diluted 1:128, diluted 1:256, diluted 1:512,diluted 1:1024, diluted 1:2048, and diluted 1:4096.

The percent of fetal DNA present in the maternal plasma was calculatedusing the following formula:

% fetal DNA=(amount of SRY gene/amount of cystic fibrosis gene)*2*100.

The amount of SRY gene was represented by the highest dilution value inwhich the gene was amplified. Likewise, the amount of cystic fibrosisgene was represented by the highest dilution value in which it wasamplified. The formula contains a multiplication factor of two (2),which is used to normalize for the fact that there is only one copy ofthe SRY gene (located on the Y chromosome), while there are two copiesof the cystic fibrosis gene.

The effect of formalin on sixty-nine (69) maternal blood samplescollected from twenty-seven clinical practices located in sixteendifferent states, spanning from Washington to Massachusetts is shown inTable XXI. In this study, formalin was added to all samples collectedfrom the pregnant women, and the percentage of fetal DNA was calculatedusing serial dilution analysis followed by PCR. The serial dilutions andPCR amplifications were performed by four different scientists over aperiod of five months. The samples were collected from women atgestational ages ranging from 11 weeks to 28 weeks, with the majority ofwomen between 16-19 weeks of gestation. A summary is provided in TableXXIII.

The average percentage of free fetal DNA for the 69 samples analyzed inthe maternal blood was 33.6%. Lo et al. reported fetal. DNAconcentrations of 3.4% in woman in late first to mid-second trimester,which was the gestational age of the majority of women in this study.Thus, the addition of formalin led to approximately a ten-fold increasein the average percentage of fetal DNA.

While the calculated percentage of fetal DNA in maternal blood isimpressive, it is also informative to examine the range of thepercentages of fetal DNA observed in this study. About six percent ofthe women (4/69) had 3.125% of free fetal DNA in the maternal blood,which was the lowest percentage of fetal. DNA observed in this study.Another 10.2% of women had 6.25% fetal DNA, which represents a two-foldincrease over the reported average in the literature. The total numberof women who had less than 10% fetal DNA in the maternal blood was only16.0%.

Fifty-eight percent of the women in this study had a percentage of fetalDNA of 25% or greater. Importantly, 26.0% of the women had fifty percentor greater fetal DNA in the maternal blood. Fetal DNA percentages ofthis magnitude have not been reported, and represent a new tool to thefield of prenatal genetics.

There were four samples collected from women at the gestational age ofeleven weeks. The percentages of fetal DNA in the maternal blood sampleswere as follows: two samples at 12.5%; one sample at 25%; and one sampleat greater than 50%. Thus, the effect of formalin on the percentages offetal DNA was observed with samples collected from women in early aswell as later gestational periods.

The effect of stabilizing cell membranes and reducing the release offree DNA was not limited to formalin. We have tested several differenttypes of agents, and combinations of agents, that prevent cell lysisand/or stabilize cell membranes, such as glutaraldehyde, and have seenthat these agents also reduce the amount of free DNA in the blood sample(data not shown).

The above described methods may also include steps of adding an agent tothe blood sample at the time or near to the time of collection tosubstantially inhibit or impede cell lysis or stabilize cell membranes.Any number of agents that impede cell lysis or stabilize cell membranesor cross-link cell membranes can be added to the maternal blood samplesincluding but not limited to formaldehyde, and derivatives offormaldehyde, formalin, glutaraldehyde, and derivatives ofglutaraldehyde, crosslinkers, primary amine reactive crosslinkers,sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfidereduction, carbohydrate reactive crosslinkers, carboxyl reactivecrosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP,APG, BASED, BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES,DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS,HBVS, sulfa-BSOCOES, Sulfa-DST, Sulfo-EGS or the compounds listed inTable XXIII. Additional cross-linkers that can be used are found at thefollowing website: www.piercenet.com/products/.

An agent that stabilizes cell membranes may be added to the maternalblood sample to reduce maternal cell lysis including but not limited toaldehydes, urea formaldehyde, phenol formaldehyde, DMAE(dimethylaminoethanol), cholesterol, cholesterol derivatives, highconcentrations of magnesium, vitamin E, and vitamin E derivatives,calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives,bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer Ahopane tetral phenylacetate, isomer B hopane tetral phenylacetate,citicoline, inositol, vitamin B, vitamin B complex, cholesterolhemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K,vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract,diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine,tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinccitrate, mexitil, dilantin, sodium hyaluronate, or polaxamer 188.

Any concentration of agent that stabilizes cell membranes, impedes celllysis or cross-link cell membranes can be added. In a preferredembodiment, the agent that stabilizes cell membranes, impedes celllysis, or cross-links cell membranes is added at a concentration thatdoes not impede or hinder subsequent reactions.

While impressive percentages of free fetal DNA in maternal blood sampleshave been reported, it is thought that higher percentages can beachieved by carefully explaining the importance of the formalin to thephysicians. Samples randomly were checked for the presence of formalinand found that about ten percent of the samples did not receiveformalin. In addition, aggregates were observed in another ten percentof the samples suggesting that the formalin had not been thoroughlymixed with the collected blood. Thus, while the addition of formalinproduced an impressive effect, it is likely that under controlledconditions, the percentage of free fetal DNA may be higher.

In addition, we believe that procedures to minimize hemolysis during thevenipuncture procedure and temperature controlled shipping containers(specimens were shipped in a Styrofoam container with ice pack, butthere was variation in temperature because samples were shipped fromvarying distances) may cause a further increase in the percentage offree fetal DNA. Needles designed to reduce hemolysis can be used duringthe venipuncture procedure.

Also, we hypothesized that procedures for carefully isolating the plasmawould help to ensure a minimal amount of maternal DNA in the sample. Weimplemented procedures, as described above, to reduce cell lysis, suchas gentle centrifugation parameters, and allowed the rotors to stopwithout external force (no brake). Also, we carefully removed thesupernatant containing the plasma DNA from the buffy-coat, whichcontains maternal DNA. These procedures coupled with the addition offormalin to prevent cell lysis resulted in a tremendous increase in thepercentage of fetal DNA.

TABLE XXI Formalin increases the percentage of free fetal DNA in bloodsamples collected at numerous clinical sites from women at variousstages of gestation. Wks Sample Gestation Fetal Genomes/ml % Fetal DNA 1 16 80 25  2 19 1066 >50  3 17 52 50  4 22 166 25  5 32 457 50  6 19400 100  7 18 800 100  8 17 100 50  9 16 50 25 10 17 25 12.5 11 16 94.7412.5 12 16 34.60 50 13 16 22.5 25 14 17 50 12.5 15 17 26.48 12.5 16 1745.00 25 17 17 94.7 100 18 17 28.13 6.25 19 19 28.13 25 20 20 11.25 12.521 15 11.25 12.5 22 11 16.66 12.5 23 18 13.23 25 24 18 12.50 6.25 25 16112.50 100 26 17 124.13 25 27 14 90.00 50 28 11 100.00 100 29 18 232.00100 30 19 626.00 100 31 19 112.50 100 32 16 423.50 100 33 16 423.50 2534 11 105.88 25 35 16 49.60 3.1 36 11 11.84 12.5 37 16 120.00 25 38 18342.90 100 39 17 51.43 25 40 18 225.00 6.25 41 17 400.00 12.5 42 28180.00 25 43 17 20.45 12.5 44 18 25.73 25 45 16 68.68 3.1 46 17 218.1825 47 15 75.00 6.25 48 16 40.58 3.1 49 17 100.00 25 50 17 14.06 12.5 5122 22.50 12.5 52 15 28.13 12.5 53 17 50.00 3.125 54 18 58.00 50 55 14100.00 25 56 16 58.08 25 57 16 13.64 12.5 58 16 25.00 6.25 59 20 45.0025 60 16 23.69 12.5 61 18 5.92 6.25 62 15 28.13 6.25 63 17 50.00 25 6364 16 360.00 50 65 16 25.00 12.5 66 16 48.65 25 67 16 47.38 12.5 68 1426.45 50 69 17 124.15 25 Average 17 131.15 33.6

TABLE XXII Formalin increases the percentage of free fetal DNA in bloodsamples collected at numerous clinical sites from women at variousstages of gestation. % Fetal DNA 3.125 6.25 12.5 25 50 Over 50% Number 47 18 22 7 11 Women (69) % 5.8 10.1 26.1 31.9 10.2 15.9

TABLE XXIII A representative list of cross-linkers that can be used toimpede maternal cell lysis. Cross-Linker Abbreviation succinimidylacetylthioacetate SATA succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate SMCC succinimidyl3-(2-pyridyldithio)propionate SPDPN-((2-pyridyldithio)ethyl)-4-azidosalicylamide PEAS; AET4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester ATFB, SE4-azido-2,3,5,6-tetrafluorobenzoic acid, STP ester, sodium salt ATFB,STP ester 4-azido-2,3,5,6-tetrafluorobenzyl amine, hydrochloridebenzophenone-4-isothiocyanate benzophenone-4-maleimide 4-benzoylbenzoicacid, succinimidyl ester Disuccinimidylsuberate DSSDithiobis(succinimidylpropionate) DSP3,3′-Dithiobis(sulfosuccinimidylpropionate) DTSSPBis[2-(sulfosuccinimdooxycarbonyloxy)ethyl]sulfone SULFO BSOCOESBis[2-(succinimdooxycarbonyloxy)ethyl]sulfone BSOCOESDisulfosuccinimdyltartrate SULFO DST Disuccinimdyltartrate DST Ethyleneglycolbis(succinimidylsuccinate) SULFO EGS Ethyleneglycolbis(sulfosuccinimidylsuccinate) EGS1,2-Di[3′-(2′-pyridyldithio)propionamido]butane DPDPBBis(sulfosuccinimdyl)suberate BSSSSuccinimdyl-4-(p-maleimidophenyl)butyrate SMPBSulfosuccinimdyl-4-(p-maleimidophenyl)butyrate SULFO SMPB3-Maleimidobenzoyl-N-hydroxysuccinimide ester MBS3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester SULFO MBSN-Succinimidyl(4-iodoacetyl)aminobenzoate SIABN-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate SULFO SIABSuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate SMCCSulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate SULFOSMCC Succinimidyl-6-[3-(2-pyridyldithio)propionamido)hexanoate NHS LCSPDP Sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido)hexanoateSULFO NHS LC SPDP N-Succinimdyl-3-(2-pyridyldithio)propionate SPDPN-Hydroxysuccinimidylbromoacetate NHS BROMOACETATEN-Hydroxysuccinimidyliodoacetate NHS IODOACETATE4-(N-Maleimidophenyl)butyric acid hydrazide hydrochloride MPBH4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid hydrazide MCCHhydrochloride m-Maleimidobenzoic acid hydrazidehydrochloride MBHN-(epsilon-Maleimidocaproyloxy)sulfosuccinimide SULFO EMCSN-(epsilon-Maleimidocaproyloxy)succinimide EMCSN-(p-Maleimidophenyl)isocyanate PMPI N-(kappa-Maleimidoundecanoic acid)hydrazide KMUHSuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6- LC SMCCamidocaproate) N-(gamma-Maleimidobutryloxy)sulfosuccinimide ester SULFOGMBS Succinimidyl-6-(beta-maleimidopropionamidohexanoate) SMPHN-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester SULFO KMUSN-(gamma-Maleimidobutyrloxy)succinimide GMBS Dimethyladipimidatehydrochloride DMA Dimethylpimelimidate hydrochloride DMPDimethylsuberimidate hydrochloride DMS Methyl-p-hydroxybenzimidatehydrochloride, 98% MHBH(Wood's Reagent) Amine ReactiveBis[sulfosuccinimidyl] suberate BS3Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone BSOCOES Disuccinimidylglutarate DSG DSP (Lomant's Reagent) 1,5-Difluoro-2,4-dinitrobenzeneDFDNB Dithiobis[succinimidylpropionate DTBPBis-[b-(4-Azidosalicylamido)ethyl]disulfide BASED Sulfhydryl ReactiveBM[PEO]₃(1,8-bis-Maleimidotriethyleneglycol BM[PEO]₃BM[PEO]₄(1,11-bis-Maleimidotetraethyleneglycol BM[PEO]₄1,4-bis-Maleimidobutane BMB 1,4 bis-Maleimidyl-2,3-dihydroxybutane BMDBBis-Maleimidohexane BMH Bis-Maleimidoethane BMOE1,4-Di-[3′-(2′-pyridyldithio)-propionamido]butane DPDPBDithio-bis-maleimidoethane DTME 1,6-Hexane-bis-vinylsulfone HBVSp-Azidobenzoyl hydrazide ABH Amine-Sulfhydryl ReactiveN-[a-Maleimidoacetoxy]succinimide ester AMAS N-[4-(p-Azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide APDPN-[β-Maleimidopropyloxy]succinimide ester BMPS N-e-Maleimidocaproic acidEMCA N-e-Maleimidocaproyloxy]succinimide ester EMCSN-[g-Maleimidobutyryloxy]succinimide ester GMBS N-k-Maleimidoundecanoicacid KMUA Succinimidyl-4-(N-Maleimidomethyl)cyclohexane-1-carboxy-(6-LC-SMCC amidocaproate Succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDPm-Maleimidobenzoyl-N-hydroxysuccinimide ester MBS Succinimidyl3-[bromoacetamido]propionate SBAP N-Succinimidyl iodoacetate SIAN-Succinimidyl[4-iodoacetyl]aminobenzoate SIAB Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate SMCC Succinimidyl4-[p-maleimidophenyl]butyrate SMPBSuccinimidyl-6-[β-maleimidopropionamido]hexanoate SMPH4-Succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene SMPTN-Succinimidyl 3-[2-pyridyldithio]-propionamido SPDPN-e-Maleimidocaproyloxy]sulfosuccinimide ester Sulfo-EMCSN-[g-Maleimidobutyryloxy]sulfosuccinimide ester Sulfo-GMBSN-[k-Maleimidoundecanoyloxy]sulfosuccinimide ester Sulfo-KMUS4-Sulfosuccinimidyl-6-methyl-a-(2- Sulfo-LC-SMPTpyridyldithio)toluamido]hexanoate Sulfosuccinimidyl6-(3′-[2-pyridyldithio]-propionamido)hexanoate Sulfo-LC-SPDPm-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester Sulfo-MBSN-Sulfosuccinimidyl[4-iodoacetyl]aminobenzoate Sulfo-SIABSulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylateSulfo-SMCC Sulfosuccinimidyl-4-(P-Maleimidophenyl) Butyrate Sulfo-SMPBAmino Groups N-5-Azido-2-nitrobenzoyloxysuccinimide ANB-NOS MethylN-succinimidyl adipate MSA N-Hydroxysuccinimidyl-4-azidosalicylic acidNHS-ASA N-Succinimidyl(4-azidophenyl)-1,3′-dithiopropionate SADPSulfosuccinimidyl 2-[7-amino-4-methylcoumarin-3-acetamido]ethyl- SAED1,3′dithiopropionate Sulfosuccinimidyl2[m-azido-o-nitrobenzamido]-ethyl-1,3′- SAND dithiopropionateN-Succinimidyl-6-[4′-azido-2′-nitrophenylamino] hexanoate SANPAHSulfosuccinimidyl-2-[p-azidosalicylamido]ethyl-1,3′-dithiopropionateSASD Sulfosuccinimidyl-[perfluoroazidobenzamido]ethyl-1,3′- SFADdithiopropionate N-Hydroxysulfosuccinimidyl-4-azidobenzoate Sulfo-HSABSulfosuccinimidyl[4-azidosalicylamido]-hexanoate Sulfo-NHS-LC-ASAN-Sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate Sulfo-SADPN-Sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino] hexanoateSulfo-SANPAH p-Azidophenyl glyoxal monohydrate APGN-β-Maleimidopropionic acid BMPA Carbohydrate Reactive-PhotoreactiveN-Succinimidyl-S-acetylthiopropionate SATP Sulfhydryl-CarbohydrateReactive 4-(4-N-Maleimidophenyl)butyric acid hydrazide hydrochlorideMPBH 3-(2-Pyridyldithio)propionyl hydrazide PDPH Sulfhydryl-carbonyl(aldehyde)/carboxyl reactive N-[β-Maleimidopropionic acid]hydrazide•TFABMPH N-e-Maleimidocaproic acid]hydrazide EMCH N-[k-Maleimidoundecanoicacid]hydrazide KMUH N-[p-Maleimidophenyl]isocyanate PMPI TFCS

Example 16

Fetal chromosomal abnormalities are determined by analyzing SNPs whereinthe maternal template DNA is homozygous and the template DNA obtainedfrom the plasma is heterozygous. Plasma that is isolated from blood of apregnant female contains both maternal template DNA and fetal templateDNA. Any number of SNP detection methods can be used to analyze thematernal and plasma DNA. Any DNA microarray may be used including butnot limited to commercially available and non-commercially availablearrays.

A DNA microarray can be designed to contain SNPs located on thechromosome or chromosomes of interest including but not limited to a DNAmicroarray containing SNPs located on chromosomes 13, 18, and 21, a DNAmicroarray containing SNPS located on chromosomes 13 and 18, a DNAmicroarray containing SNPS located on chromosomes 13 and 21, a DNAmicroarray containing SNPS located on chromosomes 18 and 21, a DNAmicroarray containing SNPS located on chromosomes 13, 18, 21, 15, 22, X,Y, a DNA microarray containing SNPS located on each of the autosomalchromosomes and each of the sex chromosomes, a DNA microarray containingSNPS located on chromosome 13, a DNA microarray containing SNPS locatedon chromosome 18, a DNA microarray containing SNPS located on chromosome21, a DNA microarray containing SNPS located on chromosome 15, a DNAmicroarray containing SNPS located on chromosome 17, a DNA microarraycontaining SNPS located on chromosome 22, a DNA microarray containingSNPS located on a single chromosome, and a DNA microarray containingSNPS located on multiple chromosomes.

In this example, SNPs are analyzed by GeneChip HuSNP Arrays fromAffymetrix, however any number of DNA arrays, including but not limitedto GeneChip arrays, GenFlex Tag arrays, Mapping 10K Array, otherAffymetrix arrays, and other DNA arrays can be used.

Collection of Blood Samples

In accordance with an IRB approved study, blood samples are collectedfrom pregnant women after informed consent is granted. Blood iscollected into 9 ml EDTA Vacuette tubes (catalog number NC9897284) and0.225 ml of 10% neutral buffered solution containing formaldehyde (4%w/v), is added to each tube, and each tube gently is inverted. The tubesare stored at 4° C. until ready for processing.

Any number of agents that impede cell lysis or stabilize cell membranesor cross-link cell membranes can be added to the tubes including but notlimited to formaldehyde, and derivatives of formaldehyde, formal in,glutaraldehyde, and derivatives of glutaraldehyde, crosslinkers, primaryamine reactive crosslinkers, sulfhydryl reactive crosslinkers,sulfhydryl addition or disulfide reduction, carbohydrate reactivecrosslinkers, carboxyl reactive crosslinkers, photoreactivecrosslinkers, cleavable crosslinkers, AEDP, APG, BASED, BM(PEO)₃,BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES, DFDNB, DMA, DMP, DMS,DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, sulfo-BSOCOES,Sulfo-DST, Sulfo-EGS or compounds listed in Table XXIII. Anyconcentration of agent that stabilizes cell membranes, impedes celllysis or cross-link cell membranes can be added. In a preferredembodiment, the agent that stabilizes cell membranes, impedes celllysis, or cross-links cell membranes is added at a concentration thatdoes not impede or hinder subsequent reactions.

An agent that stabilizes cell membranes may be added to the maternalblood sample to reduce maternal cell lysis including but not limited toaldehydes, urea formaldehyde, phenol formaldehyde, DMAE(dimethylaminoethanol), cholesterol, cholesterol derivatives, highconcentrations of magnesium, vitamin E, and vitamin E derivatives,calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives,bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer Ahopane tetral phenylacetate, isomer B hopane tetral phenylacetate,citicoline, inositol, vitamin B, vitamin B complex, cholesterolhemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K,vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract,diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine,tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinccitrate, mexitil, dilantin, sodium hyaluronate, or polaxamer 188.

Isolation of Plasma and Maternal Cells

The blood is stored at 4° C. until processing. The tubes are spun at1000 rpm for ten minutes in a centrifuge with braking power set at zero.The tubes are spun a second time at 1000 rpm for ten minutes. Thesupernatant (the plasma) of each sample is transferred to a new tube andspun at 3000 rpm for ten minutes with the brake set at zero. Thesupernatant is transferred to a new tube and stored at −80° C.Approximately two milliliters of the “buffy coat,” which containsmaternal cells, is placed into a separate tube and stored at −80° C.

Isolation of DNA

DNA is isolated from the plasma sample using the Qiagen Midi Kit forpurification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA iseluted in 100 μl of distilled water. The Qiagen Midi Kit also is used toisolate DNA from the maternal cells contained in the “buffy coat.”

Identification of Homozygous Maternal SNPs HuSNP Assay

The HuSNP assay is done as described by K. Lindblad-Toh et al. (NatureBiotechnology, Vol. 18, 1001-1005). The GeneChip® HuSNP™ Array isthought to enable whole genome surveys by simultaneously tracking nearly1,500 SNPs dispersed throughout the genome. In this example, HuSNP arrayis used as a representative Affymetrix array, and is not meant to limitthe use of other arrays including but not limited to GeneChip CYP450,and Affymetrix custom arrays that are designed to meet specific userrequirements.

PCR Amplification

Maternal DNA is assayed according to the HuSNP protocol supplied byAffymetrix. Inc. For each sample, 24 pools of primer pairs (50-100loci/pool at 50 nM each) are mixed with 5 ng of maternal DNA, 5 mMMgCl₂, 0.5 mM dNTPs, 1.25 U Amplitaq Gold (PE Biosystems, Foster City,Calif.), and the supplied buffer in 12.5 μl per pool. Samples aredenatured for 5 min at 95° C. followed by 30 cycles of 95° C. for 30 s,52° C.+0.2° C./cycle for 55 s, and 72° C. for 30 s; 5 cycles of 95° C.for 30 s, 58° C. for 55 s, and 72° C. for 30 s and a final extension of72° C. for 7 min. A 1:1000 dilution of each pool is made by adding 1 μlof the amplification product to 999 μl of ddH20. After, 2.5 μl of the1:1000 dilution is transferred to a new plate and amplified with 0.8 μMbiotinylated T7 and 0.8 μM biotinylated T3 primers, 4 mM MgCl2, 0.4 mMdNTPs, 2.5 U Taq and the supplied buffer in 25 μl for 8 min at 95° C.,followed by 40 cycles of 95° C. for 30 s, 55° C. for 90 s, and 72° C.for 30 s, and a final extension of 72° C. for 7 min. Then 1.5 μl fromeach pool is tested for amplification on a 3% agarose gel. For eachsample, the remainder of each the 24 pools is mixed and loaded on aMicrocon-10 spin column (Amicon Bioseparations, Bedford, Mass.). Samplesare concentrated by spinning the column for 20 min at 13,000g at roomtemperature and are eluted by inverting the column and centrifuging for3 min at 3,000g. Volumes are adjusted to 60 μl

A custom array can be designed using only the SNPs that are of interest.For example, a custom array may be designed that contains SNPs that arelocated on chromosomes 1, 13, 21, 18, 15, X, and Y.

Additionally, any number of SNPs can be amplified including SNPs locatedon any human chromosome including chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X or Y. Tworepresentative SNPs on chromosome 13 and two representative SNPs onchromosome 21 are chosen. The genomic location and sequence of SNPs maybe found at the SNP consortium (http://snp.cshl.org). If these SNPs arenot present on the array, different SNPs can be chosen.

SNP TSC0466917 (C/G), which is located on chromosome 13, is amplifiedusing the following primers:

Upstream Primer: 5′ CCAGCTGGTAGAACTT 3′ (SEQ ID NO: 629)Downstream Primer: 5′ CCCAATAGACCTATAG 3′ (SEQ ID NO: 630)

SNP TSC1172576 (T/A), which is located on chromosome 13, is amplifiedusing the following primers:

Upstream Primer: 5′ TAGCAGAATCTCTCAT 3′ (SEQ ID NO: 631)Downstream Primer: 5′ AGAGTATCTCATTTGTT 3′ (SEQ ID NO: 632)

SNP TSC0271628 (A/G), which is located on chromosome 21, is amplifiedusing the following primers:

Upstream Primer: 5′ AGGAAATTGTGAAGTA 3′ (SEQ ID NO: 633)Downstream Primer: 5′ TAACTCACTCACTATC 3′ (SEQ ID NO: 634)

SNP TSC0069805 (C/T), which is located on chromosome 21, is amplifiedusing the following primers:

Upstream Primer: 5′ CTGCTGAGTCATAGTC 3′ (SEQ ID NO: 635)Downstream Primer: 5′ TGTTCTTTGAATCAAC 3′ (SEQ ID NO: 636)

Hybridization to GeneChip Probe Arrays, Washing and Staining

5-30 μl of the sample (depending on the intensity of the chip lot) isdiluted in 3 M tetramethylammonium chloride (TMACl), 2 mM controloligonucleotide B1 (supplied by Affymetrix), 5×Denhardt's solution, 100μg/ml herring sperm DNA, 5 mM EDTA pH 8.0, 10 mM Tris pH7.8, and 0.01%Tween 20 in a volume of 135 μl and is denatured for 10 min at 95° C.After two minutes on ice, the samples are loaded into HuSNP chips andhybridized for 16 h at 44° C. and 40 r.p.m.

Each chip is washed and stained on the Affymetrix fluidics. Chips arewashed for two cycle's of two mixes with 6×SSPET (Bio Whitaker,Walkersville, Md.) (6×SSPE (sodium chloride, sodium phosphate, sodiumEDTA)+0.01% Triton-X-100) at 25° C., and for six cycles of five mixeswith 4×SSPET (4×SSPE+0.01% Triton X-100) at 35° C. Chips are stained for30 min at 25° C. with 50 μg/ml streptavidin-phycoerhthrin and 0.25 mg/mlbiotinylated anti-streptavidin antibody in 6×SSPE, 1×Denhardt'ssolution, and 0.01% Tween 20 in a volume of 500 μl. The chip is filledwith 6×SSPET following six washes of four mixes with 6×SSPET at 25° C.

After the hybridization, washing, and staining procedures, the HuSNPprobe arrays are scanned using the HP GeneArray Scanner (HuSNP MappingAssay Manual Affymetrix P/N 700308).

Scanning

The HuSNP probe arrays are scanned using the HP GeneArray Scanneraccording to the HuSNP Mapping Assay Manual (Affymetrix P/N 700308).Other scanners may be used including but not limited to the AlphaArray™Reader. Genotype calls are made automatically from the collectedhybridization signal intensities by the Affymetrix Microarray Suiteversion 5.0 software. Each allele of a SNP is represented by four orfive complementary probes with different locations of the SNP baseposition within the 20-nucleotide probes. Each of these probes, in turn,is paired with a probe of the same sequence except for a centralmismatch at or near a SNP position, intended to correct the fluorescencevalue for non-specific binding to the probe.

Each SNP is genotyped. SNPs located on chromosomes 13 and 21, whereinthe maternal DNA is homozygous, are analyzed with the DNA isolated fromthe plasma.

Analysis of DNA Isolated from Maternal Plasma

After the maternal DNA is analyzed and homozygous SNPs are identified,these SNPs are analyzed with the DNA isolated from the plasma. A lowcopy number of fetal genomes typically is present in the maternalplasma. To increase the copy number of the loci of interest, which arethe SNPs at which the maternal DNA is homozygous, primers are designedto anneal at approximately 130 bases upstream and 130 bases downstreamof each loci of interest. This is done to reduce statistical samplingerror that can occur when working with a low number of genomes, whichcan influence the ratio of one allele to another (see Example 11).

Design of Multiplex Primers

The primers are 12 bases in length. However, primers of any length canbe used including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36-45, 46-55, 56-65, 66-75, 76-85, 86-95, 96-105,106-115, 116-125, and greater than 125 bases. Primers are designed toanneal to both the sense strand and the antisense strand.

The maternal homozygous SNPs vary from sample to sample so definedsequences are not provided here. Primers are designed to anneal about130 bases upstream and downstream of the maternal homozygous SNPs. Theprimers are designed to terminate at the 3′ end in the dinucleotide “AA”to reduce the formation of primer-dimers. However, the primers can bedesigned to end in any of the four nucleotides and in any combination ofthe four nucleotides.

Multiplex PCR

Regions upstream and downstream of the maternal homozygous SNPs areamplified from the template genomic DNA using the polymerase chainreaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporatedherein by reference). This PCR reaction uses primers that annealapproximately 130 bases upstream and downstream of each loci ofinterest. The primers are mixed together and are used in a singlereaction to amplify the template DNA. This reaction is done to increasethe number of copies of the loci of interest, which eliminates errorgenerated from a low number of genomes.

For increased specificity, a “hot-start” PCR reaction is used. PCRreactions are performed using the HotStarTaq Master Mix Kit supplied byQIAGEN (catalog number 203443). The amount of template DNA and primerper reaction is optimized for each locus of interest. In this example,the 20 μl of plasma template DNA is used.

Two microliters of each forward and reverse primer, at concentrations of5 mM are pooled into a single microcentrifuge tube and mixed. Fourmicroliters of the primer mix is used in a total PCR reaction volume of50 μl (20 μl of template plasma DNA, 1 μl of sterile water, 4 μA ofprimer mix, and 25 μl of HotStar Taq, Twenty-five cycles of PCR areperformed. The following PCR conditions are used:

-   -   (1) 95° C. for 15 minutes;    -   (2) 95° C. for 30 second;    -   (3) 4° C. for 30 seconds;    -   (4) 37° C. for 30 seconds;    -   (5) Repeat steps 2-4 twenty-four (24) times;    -   (6) 72° C. for 10 minutes.

The temperatures and times for denaturing, annealing, and extension, areoptimized by trying various settings and using the parameters that yieldthe best results.

Other methods of genomic amplification can also be used to increase thecopy number of the loci of interest including but not limited to primerextension preamplification (PEP) (Zhang et al., PNAS, 89:5847-51, 1992),degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius, et al.,Genomics 13:718-25, 1992), strand displacement amplification using DNApolymerase from bacteriophage 29, which undergoes rolling circlereplication (Dean et al., Genomic Research 11:1095-99, 2001), multipledisplacement amplification (U.S. Pat. No. 6,124,120), REPLI-g™ WholeGenome Amplification kits, and Tagged PCR.

It is important to ensure that the region amplified (done to increasethe copy number of the fetal loci of interest) contains annealingsequences for the primers that are used with the CodeLink assay. Uponpurchase of the CodeLink array, each SNP and the primers used to amplifyeach SNP can be identified. With this knowledge, the multiplex primersare designed to encompass annealing regions for the primers in the HuSNPArray.

Purification of Fragment of Interest

The unused primers, and nucleotides are removed from the reaction byusing Qiagen MinElute PCR purification kits (Qiagen, Catalog Number28004). The reactions are performed following the manufacturer'sinstructions supplied with the columns. The DNA is eluted in 100 μl ofsterile water. 5 μl of each amplified loci is mixed together

CodeLink Assay, Washing, Staining and Scanning

The pooled DNA is assayed with the CodeLink Array as described above.Washing, staining, and scanning procedures are as described above.

Each SNP is genotyped. SNPs located on chromosomes 13 and 21, whereinthe maternal DNA is homozygous, and DNA isolated from the plasma isheterozygous are quantitated.

Quantification

The intensity of the signal for each allele at a heterozygous is SNP isquantitated. As discussed above, the expected ratio of allele 1 toallele 2 can be used to determine the presence or absence of achromosomal abnormality. If the maternal genome is homozygous at SNP X(A/A), and the plasma DNA is heterozygous at SNP X (A/G), then the Grepresents the distinct fetal signal. The ratio of G:A depends on thepercentage of fetal DNA present in the maternal blood.

For example, if the sample contains 50% fetal DNA, then the expectedratio is 0.33 (1 fetal G allele/(2 maternal A alleles+1 fetal Aallele)). This ratio should be constant for all chromosomes that arepresent in two copies. The ratio that is obtained for SNPs on chromosome13 should be the same as the ratio that is obtained for chromosome 21.

However, if the fetal genome contains an additional copy of chromosome21, then the ratio for this chromosome will deviate from the expectedratio. The expected ratio for a Trisomy condition with 50% fetal DNA inthe maternal blood is 0.25. Thus, by analyzing SNPs wherein the maternalgenome is homozygous, and the DNA that is isolated from the plasma isheterozygous, fetal chromosomal abnormalities can be detected.

This example explained the use of CodeLink Arrays, but it not intendedto limit the use of arrays. Any DNA array may be used including but notlimited to the DNA arrays listed in Table XXIII, or DNA arrays availablefrom any of the companies listed in Table XXIV.

Example 18

Fetal chromosomal abnormalities are determined by analyzing SNPs whereinthe maternal template DNA is homozygous and the template DNA obtainedfrom the plasma is heterozygous. Plasma that is isolated from blood of apregnant female contains both maternal template DNA and fetal templateDNA. Any number of SNP detection methods can be used to analyze thematernal and plasma DNA. In this example, SNPs are analyzed usingIllumina's BeadArray™ platform, available form Illumina in San Diego,Calif.

Collection of Blood Samples

In accordance with an IRB approved study, blood samples are collectedfrom pregnant women after informed consent is granted. Blood iscollected into 9 ml EDTA Vacuette tubes (catalog number NC9897284) and0.225 ml of 10% neutral buffered solution containing formaldehyde (4%w/v), is added to each tube, and each tube gently is inverted. The tubesare stored at 4° C. until ready for processing.

Any number of agents that impede cell lysis or stabilize cell membranescan be added to the tubes including but not limited to formaldehyde, andderivatives of formaldehyde, formalin, glutaraldehyde, and derivativesof glutaraldehyde, crosslinkers, primary amine reactive crosslinkers,sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfidereduction, carbohydrate reactive crosslinkers, carboxyl reactivecrosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP,APG, BASED, BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES,DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS,HBVS, sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS or compounds listed in TableXXIII. Any concentration of agent that stabilizes cell membranes orimpedes cell lysis can be added. In a preferred embodiment, the agentthat stabilizes cell membranes or impedes cell lysis is added at aconcentration that does not impede or hinder subsequent reactions.

An agent that stabilizes cell membranes may be added to the maternalblood sample to reduce maternal cell lysis including but not limited toaldehydes, urea formaldehyde, phenol formaldehyde, DMAE(dimethylaminoethanol), cholesterol, cholesterol derivatives, highconcentrations of magnesium, vitamin E, and vitamin E derivatives,calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives,bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer Ahopane tetral phenylacetate, isomer B hopane tetral phenylacetate,citicoline, inositol, vitamin B, vitamin B complex, cholesterolhemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K,vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract,diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine,tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinccitrate, dilantin, sodium hyaluronate, or polaxamer 188.

Isolation of Plasma and Maternal Cells

The blood is stored at 4° C. until processing. The tubes are spun at1000 rpm for ten minutes in a centrifuge with braking power set at zero.The tubes are spun a second time at 1000 rpm for ten minutes. Thesupernatant (the plasma) of each sample is transferred to a new tube andspun at 3000 rpm for ten minutes with the brake set at zero. Thesupernatant is transferred to a new tube and stored at −80° C.Approximately two milliliters of the “buffy coat,” which containsmaternal cells, is placed into a separate tube and stored at −80° C.

Isolation of DNA

DNA is isolated from the plasma sample using the Qiagen Midi Kit forpurification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA iseluted in 100 μl of distilled water. The Qiagen Midi Kit also is used toisolate DNA from the maternal cells contained in the “buffy coat.”

Identification of Homozygous Maternal SNPs

Illumina's BeadArray™ technology consists of a fiber optic based arraysystem that allegedly allows miniaturized, very-high throughput geneticanalysis, Illumina's 96-bundle Sentrix Array™ allegedly enable parallelprocessing of nearly 150,000 SNPs.

Fiber bundles are manufactured to contain nearly 50,000 individual,light transmitting fiber strands. Each fiber bundle is converted into anarray by first chemically etching a microscopic well at the end of eachfiber strand within a bundle, which creates up to 50,000 discretemicroscopic wells per bundle.

In a separate process, sensors are created by affixing a specific typeof molecule to the beads, each bead approximately 3 microns in diameter.For SNP analysis, a particular DNA sequence is attached to each bead ina batch. Illumina states that hundreds of thousands of molecules of thesame type coat each bead. Batches of coated beads are combined to form apool specific to the type of array desired. For SNP analysis, the arraypool allegedly uses DNA sequences that do not cross hybridize withthemselves or with known genomic DNA.

Next, the self-assembled array is created. By dipping bundles into apre-mixed bead pool, the coated beads self-assemble individually, onebead per well, on the end of each fiber in the bundle to create thearray. In Illumina's SNP genotyping array, the bead pool consists of upto 1500 sequences, which self assemble in each bundle of 50,000 fibersto create an array with an approximately thirty-fold redundancy.

The BeadArray bundles are assembled into a matrixed device, which iscalled the Array of Arrays™ platform, where each fiber bundle of thelarger array matches a well of a standardized microtiter plate.

Following array assembly, a decoding process is used to determine thebead type that resides in each fiber core. The DNA molecules aresynthesized using the Oligator™ custom DNA synthesis technology.

Illumina's SNP genotyping service using the BeadArray technology andother technologies that have sprung from the BeadArray technology areprovided at Illumina's facilities or at facilities that have received alicense to the BeadArray technology.

Maternal DNA samples are analyzed using BeadArrays that containoligonucleotide probes to SNPs. The oligonucleotide probes can be forSNPs located on any chromosome including human chromosome 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, andY. The BeadArrays are analyzed to identify SNPs, wherein the maternaltemplate DNA is homozygous. The identified homozygous SNPs are thenanalyzed using the DNA isolated from the maternal plasma.

Analysis of DNA Isolated from Maternal Plasma

After the maternal DNA is analyzed and homozygous SNPs are identified,these SNPs are analyzed with the DNA isolated from the plasma. A lowcopy number of fetal genomes typically exists in the maternal plasma. Toincrease the copy number of the loci of interest, which are the SNPs atwhich the maternal DNA is homozygous, primers are designed to anneal atapproximately 130 bases upstream and 130 bases downstream of each lociof interest. This is done to reduce statistical sampling error that canoccur when working with a low number of genomes; which can influence theratio of one allele to another (see Example 11).

Design of Multiplex Primers

The primers are 12 bases in length. However, primers of any length canbe used including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36-45, 46-55, 56-65, 66-75, 76-85, 86-95, 96-105,106-115, 116-125, and greater than 125 bases. Primers are designed toanneal to both the sense strand and the antisense strand.

The maternal homozygous SNPs vary from sample to sample so definedsequences are not provided here. Primers are designed to anneal about130 bases upstream and downstream of the maternal homozygous SNPs. Theprimers are designed to terminate at the 3′ end in the dinucleotide “AA”to reduce the formation of primer-dimers. However, the primers can bedesigned to end in any of the four nucleotides and in any combination ofthe four nucleotides.

Multiplex PCR

Regions upstream and downstream of the maternal homozygous SNPs areamplified from the template genomic DNA using the polymerase chainreaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporatedherein by reference). This PCR reaction uses primers that annealapproximately 130 bases upstream and downstream of each loci ofinterest. The primers are mixed together and are used in a singlereaction to amplify the template DNA. This reaction is done to increasethe number of copies of the loci of interest, which eliminates errorgenerated from a low number of genomes.

For increased specificity, a “hot-start” PCR reaction is used. PCRreactions are performed using the HotStarTaq Master Mix Kit supplied byQIAGEN (catalog number 203443). The amount of template DNA and primerper reaction is optimized for each locus of interest. In this example,the 20 μl of plasma template DNA is used.

Two microliters of each forward and reverse primer, at concentrations of5 mM are pooled into a single microcentrifuge tube and mixed. Fourmicroliters of the primer mix is used in a total PCR reaction volume of50 μl (20 μl of template plasma DNA, 1 μl of sterile water, 4 μl ofprimer mix, and 25 μl of HotStar Taq. Twenty-five cycles of PCR areperformed. The following PCR conditions are used:

-   -   (1) 95° C. for 15 minutes;    -   (2) 95° C. for 30 second;    -   (3) 4° C. for 30 seconds;    -   (4) 37° C. for 30 seconds;    -   (5) Repeat steps 2-4 twenty-four (24) times;    -   (6) 72° C. for 10 minutes.

The temperatures and times for denaturing, annealing, and extension, areoptimized by trying various settings and using the parameters that yieldthe best results.

Other methods of genomic amplification can also be used to increase thecopy number of the loci of interest including but not limited to primerextension preamplification (PEP) (Zhang et al., PNAS, 89:5847-51, 1992),degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius, et al.,Genomics 13:718-25, 1992), strand displacement amplification using DNApolymerase from bacteriophage 29, which undergoes rolling circlereplication (Dean et al., Genomic Research 11:1095-99, 2001), multipledisplacement amplification (U.S. Pat. No. 6,124,120), REPLI-g™ WholeGenome Amplification kits, and Tagged PCR.

It is important to ensure that the region amplified contains annealingsequences for the oligonucleotide probes in the BeadArray. Upon purchaseof the BeadArray service, each SNP and the primers used to analyze eachSNP are identified. With this knowledge, the multiplex primers aredesigned to encompass annealing regions for the primers in theBeadArray.

Purification of Fragment of Interest

The unused primers, and nucleotides are removed from the reaction byusing Qiagen MinElute PCR purification kits (Qiagen, Catalog Number28004). The reactions are performed following the manufacturer'sinstructions supplied with the columns. The DNA is eluted in 100 μl ofsterile water. 5 μl of each amplified loci is mixed together

BeadArray Technology

The pooled DNA is assayed with the BeadArray as described above. EachSNP is genotyped. SNPs located on chromosomes 13 and 21, wherein thematernal DNA is homozygous, and DNA isolated from the plasma isheterozygous are quantitated.

Quantification

The intensity of the signal for each allele at a heterozygous is SNP isquantitated. As discussed above, the expected ratio of allele 1 toallele 2 can be used to determine the presence or absence of achromosomal abnormality. If the maternal genome is homozygous at SNP X(A/A), and the plasma DNA is heterozygous at SNP X (A/G), then the Grepresents the distinct fetal signal. The ratio of G:A depends on thepercentage of fetal DNA present in the maternal blood.

For example, if the sample contains 50% fetal DNA, then the expectedratio is 0.33 (1 fetal G allele/(2 maternal A alleles+1 fetal Aallele)). This ratio should be constant for all chromosomes that arepresent in two copies. The ratio that is obtained for SNPs on chromosome13 should be the same as the ratio that is obtained for chromosome 21.

However, if the fetal genome contains an additional copy of chromosome21, then the ratio for this chromosome will deviate from the expectedratio. The expected ratio for a Trisomy condition with 50% fetal DNA inthe maternal blood is 0.25. Thus, by analyzing SNPs wherein the maternalgenome is homozygous, and the DNA that is isolated from the plasma isheterozygous, fetal chromosomal abnormalities can be detected.

This example explained the use of Illumines BeadArray Technology, but itnot intended to limit the use of arrays. Any DNA array may be usedincluding but not limited to the DNA arrays listed in Table XXIII, orDNA arrays available from any of the companies listed in Table XXIV.

Example 19

Fetal chromosomal abnormalities are determined by analyzing SNPs whereinthe maternal template DNA is homozygous and the template DNA obtainedfrom the plasma is heterozygous. Plasma that is isolated from blood of apregnant female contains both maternal template DNA and fetal templateDNA. Any number of SNP detection methods can be used to analyze thematernal and plasma DNA. In this example, SNPs are analyzed usingSequenom's MassArray™ System, which uses Sequenom's homogenousMassCleave™ (hMC) method.

Collection of Blood Samples

In accordance with an IRB approved study, blood samples are collectedfrom pregnant women after informed consent is granted. Blood iscollected into 9 ml EDTA Vacuette tubes (catalog number NC9897284) and0.225 ml of 10% neutral buffered solution containing formaldehyde (4%w/v), is added to each tube, and each tube gently is inverted. The tubesare stored at 4° C. until ready for processing.

Any number of agents that impede cell lysis or stabilize cell membranescan be added to the tubes including but not limited to formaldehyde, andderivatives of formaldehyde, formalin, glutaraldehyde, and derivativesof glutaraldehyde, crosslinkers, primary amine reactive crosslinkers,sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfidereduction, carbohydrate reactive crosslinkers, carboxyl reactivecrosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP,APG, BASED, BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES,DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS,HBVS, sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS or compounds listed in TableXXIII. Any concentration of agent that stabilizes cell membranes orimpedes cell lysis can be added. In a preferred embodiment, the agentthat stabilizes cell membranes or impedes cell lysis is added at aconcentration that does not impede or hinder subsequent reactions.

An agent that stabilizes cell membranes may be added to the maternalblood sample to reduce maternal cell lysis including but not limited toaldehydes, urea formaldehyde, phenol formaldehyde, DMAE(dimethylaminoethanol), cholesterol, cholesterol derivatives, highconcentrations of magnesium, vitamin E, and vitamin E derivatives,calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives,bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer Ahopane tetral phenylacetate, isomer B hopane tetral phenylacetate,citicoline, inositol, vitamin B, vitamin B complex, cholesterolhemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K,vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract,diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine,tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinccitrate, mexitil, dilantin, sodium hyaluronate, or polaxamer 188.

Isolation of Plasma and Maternal Cells

The blood is stored at 4° C. until processing. The tubes are spun at1000 rpm for ten minutes in a centrifuge with braking power set at zero.The tubes are spun a second time at 1000 rpm for ten minutes. Thesupernatant (the plasma) of each sample is transferred to a new tube andspun at 3000 rpm for ten minutes with the brake set at zero. Thesupernatant is transferred to a new tube and stored at −80° C.Approximately two milliliters of the “buffy coat,” which containsmaternal cells, is placed into a separate tube and stored at −80° C.

Isolation of DNA

DNA is isolated from the plasma sample using the Qiagen Midi Kit forpurification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA iseluted in 100 μl of distilled water. The Qiagen Midi Kit also is used toisolate DNA from the maternal cells contained in the “buffy coat.”

Identification of Homozygous Maternal SNPs Targeted SNP Discovery: hMCMethod

Sequenom's hMC method uses nucleotide base-specific cleavage forgenotyping. The cleaved fragments are measured using MALDI-TOF togenerate a characteristic peak signal, based on the mass of eachfragment, for any particular sequence.

Primer Design

Four primers are needed for the two PCR reactions (one forward reactionand one reverse reaction). The recommended size range for PCR ampliconsis 300-700 base pairs. The primers contain a T-7 promoter tagged forwardor reverse primer to obtain an appropriate product for in vitrotranscription. An 8 base insert is included to prevent abortive cycling.The primer that lacks the T-7 promoter contains a 10-mer tag in order tobalance the primers.

The primers for one SNP are provided below. SNP TSC1172576 (T/A), whichis located on chromosome 13, is amplified using the following primersfor the forward reaction:

Forward Reaction:

Upstream Primer: (SEQ ID NO: 637) 5′ CAGTAATACGACTCACTATAGGGGTCAGGATTAGCAGAATCTCTC AT 3′ Downstream Primer: (SEQ ID NO: 638)5′ GCATTCTATGAGAGTATCTCATTTGTT 3′

Reverse Reaction:

Upstream Primer (SEQ ID NO: 639) 5′ CAGTAATACGACTCACTATAGGGGTCAGGAAGAGTATCTCATTTGT T 3′ Downstream Primer (SEQ ID NO: 640)5′ GCATTCTATGTAGCAGAATCTCTCAT 3′

The sequence of the T-7 promoter are in italics, the 8 base insert isunderlined, the 10-base balancing sequence is double underlined, and thegene specific sequences are unmodified.

PCR Amplification

Five nanograms of DNA is amplified in a 5 μl volume using a384-microtiter format. The following PCR conditions are used:

1) 94° C. for 15 minutes;

2) 94° C. for 20 seconds;

3) 62° C. for 30 seconds;

4) 72° C. for 1 minute;

5) Repeat steps 2-4 44 times; and

6) 72° C. for 3 minutes.

Dephosphorylation

Shrimp Alkaline Phosphatase (SAP) (2 μl) is added to each 5 μl PCRreaction to dephosphorylate unincorporated dNTPs from the PCR reaction.The plates are incubated at 37° C. for 20 minutes. Then, the plates areincubated at 85° C. for 5 minutes.

In Vitro Transcription

For each transcription reaction, 2 μl of transcription cocktail and 2 μlof PCR/SAP sample are needed. Add 2 μl of transcription cocktail and 2μl of PCR/SAP sample to a new microtiter plate. The plates are incubatedat 37° C. for two hours. For detailed information regarding theseprotocols see the “Processing homogeneous MassCLEAVE Reactions” chapterin the MassARRAY Liquid Handler SNP Discovery User's Guide forinstructions, which is fully incorporated herein by reference,

RNase A Cleavage

RNase A cocktail (2.5 μl) is added to each reaction (T cleavage and Ccleavage). The plates are incubated at 37° C. for one hour.

Depending on the nucleotide at the SNP site, various fragments ofdifferent weights are generated. For example, the DNA sequencesurrounding SNP TSC1172576, which is located on chromosome 13, is asfollows:

5′ CCGCATA T/A CTCAGCACA 3′ (SEQ ID NO: 641) 3′GGCGTAT A/T GAGTCGTGT 5′(SEQ ID NO: 642)

After PCR, in vitro transcription, and base-specific cleavage, thefollowing fragments for each allele are generated:

T allele A allele Products of forward 1) TATCTCA 1) CTCAGC Transcription2) ATC 2) AAC Products of reverse 1) AGTTA 1) AGATA transcription 2)GTTATGC 2) ATATGCG

For the ATC and AAC fragments, the weight difference between T and A isused to determine the genotype at SNP TSC1172576. Likewise, the weightdifference between T and A in fragments AGTTA and AGATA is used todetermine the genotype at SNP TSC1172576.

Sample Conditioning

Double distilled water (20 μl) is added to each sample within the384-well plate. Clean Resin (6 mg) is added to each well. The plate isrotated for 10 minutes, followed by a centrifugation at 3200×g. It isrecommended that water always be added before the Clean Resin.

Sample Transfer

The hMC reaction product (10-15 μl is dispensed onto a 384 elementSpectroCHIP®. For further information, see the “Dispensing MassCLEAVEReaction Products onto SpectroCHIPs” chapter in the MassARRAYNanodispenser SNP Discovery User's Guide for instructions.

Sample Analysis

Spectra from the four cleavage reactions is acquired using theMassARRAY™ system. For further instructions, see the “Acquiring Spectra”chapter in the MassARRAY Discovery RT Software User's guide forinstructions on acquiring spectra from SpectroCHIPS®.

SNP Analysis

The results are analyzed using the SNP Discovery Analysis software. Forfurther instructions, see the “Analyzing SNPs” chapter in the MassARRAYDiscovery RT Software User's Guide for instructions on using the SNPDiscovery Analysis software. Components that are useful for theMassARRAY procedure include MassARRAY™ Analyzer (part number 004500),MassARRAY™ Discovery RT Software version 1.2 (part number 11434),MassARRAY™ SNP Discovery Starter Kit (part number 10027), and LiquidHandler SNP Discovery Methods and Macros (part number 11433).

The SpectroCHIP array is used to genotype the maternal DNA following themanufacturer's recommended protocols and procedures, which are madeavailable after purchase of the SpectroCHIP array. SNPs at which thematernal DNA is homozygous are used to analyze the DNA isolated from thematernal plasma.

Analysis of DNA Isolated from Maternal Plasma

After the maternal DNA is analyzed and homozygous SNPs are identified,these SNPs are analyzed with the DNA isolated from the plasma. A lowcopy number of fetal genomes typically exists in the maternal plasma. Toincrease the copy number of the loci of interest, which are the SNPs atwhich the maternal DNA is homozygous, primers are designed to anneal atapproximately 130 bases upstream and 130 bases downstream of each lociof interest. This is done to reduce statistical sampling error that canoccur when working with a low number of genomes, which can influence theratio of one allele to another (see Example 11).

Design of Multiplex Primers

The primers are 12 bases in length. However, primers of any length canbe used including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36-45, 46-55, 56-65, 66-75, 76-85, 86-95, 96-105,106-115, 116-125, and greater than 125 bases. Primers are designed toanneal to both the sense strand and the antisense strand.

The maternal homozygous SNPs vary from sample to sample so definedsequences are not provided here. Primers are designed to anneal about130 bases upstream and downstream of the maternal homozygous SNPs. Theprimers are designed to terminate at the 3′ end in the dinucleotide “AA”to reduce the formation of primer-dimers. However, the primers can bedesigned to end in any of the four nucleotides and in any combination ofthe four nucleotides.

Multiplex PCR

Regions upstream and downstream of the maternal homozygous SNPs areamplified from the template genomic DNA using the polymerase chainreaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporatedherein by reference). This PCR reaction uses primers that annealapproximately 130 bases upstream and downstream of each loci ofinterest. The primers are mixed together and are used in a singlereaction to amplify the template DNA. This reaction is done to increasethe number of copies of the loci of interest, which eliminates errorgenerated from a low number of genomes.

For increased specificity, a “hot-start” PCR reaction is used. PCRreactions are performed using the HotStarTaq Master Mix Kit supplied byQIAGEN (catalog number 203443). The amount of template DNA and primerper reaction is optimized for each locus of interest. In this example,the 20 μl of plasma template DNA is used.

Two microliters of each forward and reverse primer, at concentrations of5 mM are pooled into a single microcentrifuge tube and mixed. Fourmicroliters of the primer mix is used in a total PCR reaction volume of50 μl (20 μl of template plasma DNA, 1 μl of sterile water, 4 μl ofprimer mix, and 25 μl of HotStar Taq. Twenty-five cycles of PCR areperformed. The following PCR conditions are used:

-   -   (1) 95° C. for 15 minutes;    -   (2) 95° C. for 30 second;    -   (3) 4° C. for 30 seconds;    -   (4) 37° C. for 30 seconds;    -   (5) Repeat steps 2-4 twenty-four (24) times;    -   (6) 72° C. for 10 minutes.

The temperatures and times for denaturing, annealing, and extension, areoptimized by trying various settings and using the parameters that yieldthe best results.

Other methods of genomic amplification can also be used to increase thecopy number of the loci of interest including but not limited to primerextension preamplification (PEP) (Zhang et al., PNAS, 89:5847-51, 1992),degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius, et al.,Genomics 13:718-25, 1992), strand displacement amplification using DNApolymerase from bacteriophage 29, which undergoes rolling circlereplication (Dean et al., Genomic Research 11:1095-99, 2001), multipledisplacement amplification (U.S. Pat. No. 6,124,120), REPLI-g™ WholeGenome Amplification kits, and Tagged PCR.

It is important to ensure that the region amplified contains annealingsequences for the PCR primers in the targeted SNP discovery, hMC method

Purification of Fragment of Interest

The unused primers, and nucleotides are removed from the reaction byusing Qiagen MinElute PCR purification kits (Qiagen, Catalog Number28004). The reactions are performed following the manufacturer'sinstructions supplied with the columns. The DNA is eluted in 100 μl ofsterile water. 5 μl of each amplified loci is mixed together

Targeted SNP Discovery: hMC Method

The pooled DNA is assayed with the INC method as described above. EachSNP is genotyped. SNPs located on chromosomes 13 and 21, wherein thematernal DNA is homozygous, and DNA isolated from the plasma isheterozygous are quantitated. However, SNPs located on other chromosomescan also be quantitated if so desired.

Quantification

The intensity of each peak, wherein each peak corresponds to a DNAfragment with a specific molecular weight, is quantitated. As discussedabove, the expected ratio of allele 1 to allele 2 is used to determinethe presence or absence of a chromosomal abnormality. If the maternalgenome is homozygous at SNP X (NA), and the plasma DNA is heterozygousat SNP X (A/G), then the G represents the distinct fetal signal.

There will be some fragments that differ in molecular due to thepresence of the G nucleotide at SNP X in the fetal genome. The intensityof the peak with the A nucleotide is quantitated and the intensity ofthe peak that corresponds to fragments with the G nucleotide isquantitated. The ratio of G:A depends on the percentage of fetal DNApresent in the maternal blood.

For example, if the sample contains 50% fetal. DNA, then the expectedratio is 0.33 (1 fetal U allele/(2 maternal A alleles+1 fetal Aallele)). This ratio should be constant for all chromosomes that arepresent in two copies. The ratio that is obtained for SNPs on chromosome13 should be the same as the ratio that is obtained for chromosome 21.

However, if the fetal genome contains an additional copy of chromosome21, then the ratio for this chromosome will deviate from the expectedratio. The expected ratio for a Trisomy condition with 50% fetal DNA inthe maternal blood is 0.25. Thus, by analyzing SNPs wherein the maternalgenome is homozygous, and the DNA that is isolated from the plasma isheterozygous, fetal chromosomal abnormalities can be detected.

This example explained the use of Sequenom's hMC method, but it notintended to limit the use of other mass spectrometry techniques.

Example 20

Fetal chromosomal abnormalities are determined by analyzing SNPs whereinthe maternal template DNA is homozygous and the template DNA obtainedfrom the plasma is heterozygous. Plasma that is isolated from blood of apregnant female contains both maternal template DNA and fetal templateDNA. Any number of SNP detection methods can be used to analyze thematernal and plasma DNA. In this example, SNPs are analyzed usingSequenom's MassArray™ Homogenous MassEXTENDT™ (hME) Assay.

Collection of Blood Samples

In accordance with an IRB approved study, blood samples are collectedfrom pregnant women after informed consent is granted. Blood iscollected into 9 ml EDTA Vacuette tubes (catalog number NC9897284) and0.225 ml of 10% neutral buffered solution containing formaldehyde (4%w/v), is added to each tube, and each tube gently is inverted. The tubesare stored at 4° C. until ready for processing.

Any number of agents that impede cell lysis or stabilize cell membranescan be added to the tubes including but not limited to formaldehyde, andderivatives of formaldehyde, formalin, glutaraldehyde, and derivativesof glutaraldehyde, crosslinkers, primary amine reactive crosslinkers,sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfidereduction, carbohydrate reactive crosslinkers, carboxyl reactivecrosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP,APG, BASED, BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES,DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS,HBVS, sulfo-BSOCOES, Sulfo-DST, Sulfa-EGS or compounds listed in TableXXIII. Any concentration of agent that stabilizes cell membranes orimpedes cell lysis can be added. In a preferred embodiment, the agentthat stabilizes cell membranes or impedes cell lysis is added at aconcentration that does not impede or hinder subsequent reactions.

An agent that stabilizes cell membranes may be added to the maternalblood sample to reduce maternal cell lysis including but not limited toaldehydes, urea formaldehyde, phenol formaldehyde, DMAE(dimethylaminoethanol), cholesterol, cholesterol derivatives, highconcentrations of magnesium, vitamin E, and vitamin E derivatives,calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives,bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer Ahopane tetral phenylacetate, isomer B hopane tetral phenylacetate,citicoline, inositol, vitamin B, vitamin B complex, cholesterolhemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K,vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract,diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine,tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinccitrate, mexitil, dilantin, sodium hyaluronate, or polaxamer 188.

Isolation of Plasma and Maternal Cells

The blood is stored at 4° C. until processing. The tubes are spun at1000 rpm for ten minutes in a centrifuge with braking power set at zero.The tubes are spun a second time at 1000 rpm for ten minutes. Thesupernatant (the plasma) of each sample is transferred to a new tube andspun at 3000 rpm for ten minutes with the brake set at zero. Thesupernatant is transferred to a new tube and stored at −80° C.Approximately two milliliters of the “buffy coat,” which containsmaternal cells, is placed into a separate tube and stored at −80° C.

Isolation of DNA

DNA is isolated from the plasma sample using the Qiagen Midi Kit forpurification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA iseluted in 100 μl of distilled water. The Qiagen Midi Kit also is used toisolate DNA from the maternal cells contained in the “buffy coat.”

Identification of Homozygous Maternal SNPs

MassARRAY Homogenous MassEXTEND™ (hME) Assay

The Homogenous MassEXTEND™ (hME) Assay uses a beadless, label-freeprimer extension chemistry for genotyping. Each of the primer productshas a unique molecular weight that allows the associated genotype to beprecisely identified using mass spectrometry.

Template Amplification

The isolated maternal DNA is amplified (2.5 ng) in a 5 μl volume using a384-microtiter plate format. Any number of SNPs can be amplified, eitherin a single reaction or in multiple reactions. Representative primersthat are used to amplify SNP TSC0271628 (A/G), which is located onchromosome 21, are provided below:

Upstream Primer: 5′ AGGAAATTGTGAAGTA 3′ (SEQ ID NO: 643) DownstreamPrimer: 5′ TAACTCACTCACTATC 3′ (SEQ ID NO: 644)

The primers can be longer or shorter in nucleotide sequence. PCRconditions recommended by the makers of MassARRAY Homogenous MassEXTENDAssay are followed. Representative PCR conditions are provided below:

-   -   (1) 95° C. for 15 minutes and 15 seconds;    -   (2) 95° C. for 30 seconds;    -   (4) 57° C. for 30 seconds;    -   (5) 72° C. for 30 seconds;    -   (6) Repeat steps 2-5 thirty two (32) times;    -   (7) 72° C. for 5 minutes.

Dephosphorylation

Arctic shrimp alkaline phosphatase is added to the samples, which arethen incubated at 37° C. for 20 minutes. This step is done todephosphorylate any remaining nucleotides, which prevents their futureincorporation and interference with MassARRAY Homogenous MassEXTENDAssay. Samples are then incubated at 85° C. to inactivate theheat-labile SAP.

hME Reaction

A MassEXTEND primer is designed to anneal close to the polymorphic site,and is designed to identify both alleles of the polymorphic site. ForSNP TSC0271628, a representative MassEXTEND primer is:

5′ CTTTTTATGCCTTTCCACTCATCCA 3′ (SEQ ID NO: 645)

The length of the MassEXTEND primer is designed according to theinstructions provided by the makers of the MassARRAY HomogenousMassEXTEND Assay.

The MassEXTEND primer, DNA polymerase, and a cocktail mixture ofdeoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs) are added tothe initial primer extension reaction. Allele-specific primer productsare generated that are generally one to four bases longer than theoriginal MassEXTEND primer.

A MassEXTEND primer is hybridized closely adjacent to the polymorphicsite following the conditions recommended by the makers of the MassARRAYHomogenous MassEXTEND Assay. Nucleotide mixtures are selected tomaximize mass differences for all possible MassEXTEND products.Appropriate dNTPS are incorporated until a single ddNTP is incorporated,and the reaction is terminated. The manufacturer's protocols arefollowed for all steps of the hME assay.

Representative reaction products for SNP TSC0271628 are provided below:

A Allele Before Primer Extension

(SEQ ID NO: 646) MassEXTEND primer: CT TTTT ATGCCT T TCCACTCATCCA (SEQID NO: 647) Sample DNA: GAAAAATACGGAAAGGTGAGTAGGTTTCC

The SNP site is identified in bold. After incubation with DNApolymerase, ddATP, dCTP, dGTP, and dTTP, the following product isgenerated:

A Allele after Primer Extension

(SEQ ID NO: 648) MassEXTEND primer: CT TTTT ATGCCT T TCCACTCATCCAA* (SEQID NO: 649) Sample DNA: GAAAAATACGGAAAGGTGAGTAGGTTTCC

ddATP is incorporated into the primer. Either labeled or unlabeledddNTPs can be used. The asterisk indicates ddATP that is unlabeled.After the incorporation reaction, a 24-mer primer is generated.

G Allele Before Primer Extension

(SEQ ID NO: 650) MassEXTEND primer: CT TTTT ATGCCT T TCCACTCATCCA (SEQID NO: 651) Sample DNA: GAAAAATACGGAAAGGTGAGTAGGTCTCC

The SNP site is identified in bold. After incubation with DNApolymerase, ddATP, dCTP, dGTP, and dTTP, the following product isgenerated:

G Allele after Primer Extension

(SEQ ID NO: 652) MassEXTEND primer: CT TTTT ATGCCT T TCCACTCATCCAG A*(SEQ ID NO: 653) Sample DNA: GAAAAATACGGAAAGGTGAGTAGGTCTCC

After the incorporation reaction, a 25-mer primer is generated. Thedifference in molecular weight between the reaction product for the Aallele (24-mer) and the reaction product for the G allele (25-mer) isused to genotype the locus of interest.

Sample Conditioning

SpectroCLEAN™ resin is added to the reaction to remove extraneous saltsthat interfere with MALDI-TOF analysis.

Sample Transfer

15 nl of sample is transferred from the 384-microtiter plate and spottedonto the pad of the 384 SpectroCHIP™ microarray.

Sample Analysis

The SpectroCHIP™ is placed into the MALDI-TOF, which measures the massof the extension products. Once determined, the genotype is called inreal-time with SpectroTYPER™ RT software. SNPs at which the maternal DNAare homozygous are identified, and analyzed with the DNA that isisolated from the plasma.

Analysis of DNA Isolated from Maternal Plasma

After the maternal DNA is analyzed and homozygous SNPs are identified,these SNPs are analyzed with the DNA isolated from the plasma. A lowcopy number of fetal genomes typically exists in the maternal plasma. Toincrease the copy number of the loci of interest, which are the SNPs atwhich the maternal DNA is homozygous, primers are designed to anneal atapproximately 130 bases upstream and 130 bases downstream of each lociof interest. This is done to reduce statistical sampling error that canoccur when working with a low number of genomes, which can influence theratio of one allele to another (see Example 11).

Design of Multiplex Primers

The primers are 12 bases in length. However, primers of any length canbe used including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36-45, 46-55, 56-65, 66-75, 76-85, 86-95, 96-105,106-115, 116-125, and greater than 125 bases. Primers are designed toanneal to both the sense strand and the antisense strand.

The maternal homozygous SNPs vary from sample to sample so definedsequences are not provided here. Primers are designed to anneal about130 bases upstream and downstream of the maternal homozygous SNPs. Theprimers are designed to terminate at the 3′ end in the dinucleotide “AA”to reduce the formation of primer-dimers. However, the primers can bedesigned to end in any of the four nucleotides and in any combination ofthe four nucleotides.

Multiplex PCR

Regions upstream and downstream of the maternal homozygous SNPs areamplified from the template genomic DNA using the polymerase chainreaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporatedherein by reference). This PCR reaction uses primers that annealapproximately 130 bases upstream and downstream of each loci ofinterest. The primers are mixed together and are used in a singlereaction to amplify the template DNA. This reaction is done to increasethe number of copies of the loci of interest, which eliminates errorgenerated from a low number of genomes.

For increased specificity, a “hot-start” PCR reaction is used. PCRreactions are performed using the HotStarTaq Master Mix Kit supplied byQIAGEN (catalog number 203443). The amount of template DNA and primerper reaction is optimized for each locus of interest. In this example,the 20 μl of plasma template DNA is used.

Two microliters of each forward and reverse primer, at concentrations of5 mM are pooled into a single microcentrifuge tube and mixed. Fourmicroliters of the primer mix is used in a total PCR reaction volume of50 μl (20 μl of template plasma DNA, 1 μl of sterile water, 4 μl ofprimer mix, and 25 μl of HotStar Taq. Twenty-five cycles of PCR areperformed. The following PCR conditions are used:

-   -   (1) 95° C. for 15 minutes;    -   (2) 95° C. for 30 second;    -   (3) 4° C. for 30 seconds;    -   (4) 37° C. for 30 seconds;    -   (5) Repeat steps 2-4 twenty-four (24) times;    -   (6) 72° C. for 10 minutes.

The temperatures and times for denaturing, annealing, and extension, areoptimized by trying various settings and using the parameters that yieldthe best results.

Other methods of genomic amplification can also be used to increase thecopy number of the loci of interest including but not limited to primerextension preamplification (PEP) (Zhang et al., PNAS, 89:5847-51, 1992),degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius, et al.,Genomics 13:718-25, 1992), strand displacement amplification using DNApolymerase from bacteriophage 29, which undergoes rolling circlereplication (Dean et al., Genomic Research 11:1095-99, 2001), multipledisplacement amplification (U.S. Pat. No. 6,124,120), REPLI-g™ WholeGenome Amplification kits, and Tagged PCR.

Purification of Fragment of Interest

The unused primers, and nucleotides are removed from the reaction byusing Qiagen MinElute PCR purification kits (Qiagen, Catalog Number28004). The reactions are performed following the manufacturer'sinstructions supplied with the columns. The DNA is eluted in 100 μl ofsterile water. 5 μl of each amplified loci is mixed together

MassARRAY Homogenous MassEXTEND Assay

The pooled DNA is assayed with the hME assay as described above. EachSNP is genotyped. SNPs located on chromosomes 13 and 21, wherein thematernal DNA is homozygous, and DNA isolated from the plasma isheterozygous are quantitated.

Quantification

The intensity of each peak, wherein each peak corresponds to a DNAfragment with a specific molecular weight, is quantitated. As discussedabove, the expected ratio of allele 1 to allele 2 is used to determinethe presence or absence of a chromosomal abnormality. If the maternalgenome is homozygous at SNP X (A/A), and the plasma DNA is heterozygousat SNP X (A/G), then the G represents the distinct fetal signal.

There will be some fragments that differ in molecular due to thepresence of the G nucleotide at SNP X in the fetal genome. The intensityof the peak with the A nucleotide is quantitated and the intensity ofthe peak that corresponds to fragments with the G nucleotide isquantitated. The ratio of G:A depends on the percentage of fetal DNApresent in the maternal blood.

For example, if the sample contains 50% fetal DNA, then the expectedratio is 0.33 (1 fetal G allele/(2 maternal A alleles+1 fetal Aallele)). This ratio should be constant for all chromosomes that arepresent in two copies. The ratio that is obtained for SNPs on chromosome13 should be the same as the ratio that is obtained for chromosome 21.

However, if the fetal genome contains an additional copy of chromosome21, then the ratio for this chromosome will deviate from the expectedratio. The expected ratio for a Trisomy condition with 50% fetal DNA inthe maternal blood is 0.25. Thus, by analyzing SNPs wherein the maternalgenome is homozygous, and the DNA that is isolated from the plasma isheterozygous, fetal chromosomal abnormalities can be detected.

This example explained the use Sequenom's MassARRAY HomogenousMassEXTEND (hME) assay, but it not intended to limit the use oftechniques that differentiate molecules based on molecular weight.

Example 21

Fetal chromosomal abnormalities are determined by analyzing SNPs whereinthe maternal template DNA is homozygous and the template DNA obtainedfrom the plasma is heterozygous. Plasma that is isolated from blood of apregnant female contains both maternal template DNA and fetal templateDNA. Any number of SNP detection methods can be used to analyze thematernal and plasma DNA. In this example, SNPs are analyzed usingOrchid's SNP-IT™ Assay. However, other SNP detection methods based onprimer extension may also be used.

Collection of Blood Samples

In accordance with an IRB approved study, blood samples are collectedfrom pregnant women after informed consent is granted. Blood iscollected into 9 ml EDTA Vacuette tubes (catalog number NC9897284) and0.225 ml of 10% neutral buffered solution containing formaldehyde (4%w/v), is added to each tube, and each tube gently is inverted. The tubesare stored at 4° C. until ready for processing.

Any number of agents that impede cell lysis or stabilize cell membranescan be added to the tubes including but not limited to formaldehyde, andderivatives of formaldehyde, formalin, glutaraldehyde, and derivativesof glutaraldehyde, crosslinkers, primary amine reactive crosslinkers,sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfidereduction, carbohydrate reactive crosslinkers, carboxyl reactivecrosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP,APG, BASED, BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES,DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS,HBVS, sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS or compounds listed in TableXXIII. Any concentration of agent that stabilizes cell membranes orimpedes cell lysis can be added. In a preferred embodiment, the agentthat stabilizes cell membranes or impedes cell lysis is added at aconcentration that does not impede or hinder subsequent reactions.

An agent that stabilizes cell membranes may be added to the maternalblood sample to reduce maternal cell lysis including but not limited toaldehydes, urea formaldehyde, phenol formaldehyde, DMAE(dimethylaminoethanol), cholesterol, cholesterol derivatives, highconcentrations of magnesium, vitamin E, and vitamin E derivatives,calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives,bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer Ahopane tetral phenylacetate, isomer B hopane tetral phenylacetate,citicoline, inositol, vitamin B, vitamin B complex, cholesterolhemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K,vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract,diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine,tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinccitrate, mexitil, dilantin, sodium hyaluronate, or polaxamer 188.

Isolation of Plasma and Maternal Cells

The blood is stored at 4° C. until processing. The tubes are spun at1000 rpm for ten minutes in a centrifuge with braking power set at zero.The tubes are spun a second time at 1000 rpm for ten minutes. Thesupernatant (the plasma) of each sample is transferred to a new tube andspun at 3000 rpm for ten minutes with the brake set at zero. Thesupernatant is transferred to a new tube and stored at −80° C.Approximately two milliliters of the “bully coat,” which containsmaternal cells, is placed into a separate tube and stored at −80° C.

Isolation of DNA

DNA is isolated from the plasma sample using the Qiagen Midi Kit forpurification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA iseluted in 100 μl of distilled water. The Qiagen Midi Kit also is used toisolate DNA from the maternal cells contained in the “buffy coat.”

Identification of Homozygous Maternal SNPs SNP-IT™ Assay

The SNP-IT Assay is based on a single base primer extension. Prior tothe SNP-IT Assay, a PCR product that includes the SNP of interest isprepared, using one unmodified and one phosphorothiolate modifiedprimer. The PCR product is then rendered single stranded usingexonuclease, and the single stranded DNA is annealed to a SNP-IToligonucleotide immobilized on the surface of a 96-well microtiterplate. Following hybridization, single base extension occurs by theaddition of DNA polymerase and the labeled terminators. The incorporatedbase is detected using antibodies specific to the label followed bycolorimetric detection. Data analysis can be done visually or with theuse of the absorbance plate reader.

Primer Design

For each locus of interest, the SNP-IT™ Assay requires three primers.The primers are designed to produce an amplicon of 100-50 base pairs.The sequence of the SNP-IT primer, which is designed to annealimmediately upstream of the SNP site, is the best sequence availablefrom between the upper and the lower strands. The sequence of the SNP-ITprimer is designed to minimize hybridization to self and other sites inthe amplicon. In addition, the SNP-IT primer may contain modified basesto prevent self-priming. The length of the primers is designed accordingto the makers of the SNP-IT Assay.

Representative primers for the amplification and genotyping of SNPTSC0069085, which is located on chromosome 21, are provided below:

Upstream Primer: 5′ ATCACACTGGGGATC 3′ (SEQ ID NO: 654) DownstreamPrimer: 5′ CTAAACCTATGACTC 3′ (SEQ ID NO: 655) SNP-IT primer5′ TTCACAGAGGATATCTTAATA 3′ (SEQ ID NO: 656)

The upstream primer is unmodified and the downstream primer isphosphorothiolate modified.

SNP-IT Plate Coating

SNP-IT primer is added to coat wells of empty 96-well plates. Thisreaction typically incubates overnight. The manufacturer's protocols andprocedures are followed.

PCR

Template DNA (15 ng) is amplified either in a reaction vessel includingbut not limited to an eppendorf tube or a well of a microtiter plate.The manufacturer's protocols and procedures are followed for the PCRreaction.

Exonuclease

PCR product is treated with exonuclease to degrade the unmodifiedstrand. The protected phosphorothiolate-labeled strand is used in theSNP-IT Assay. The manufacturer's protocols and procedures are followedfor the exonuclease reaction.

Annealing

Single stranded PCR product transferred to SNP-IT plate and is allowedto form a hybrid with the SNP-IT primer. The annealing reactiontypically proceeds for one hour. The manufacturer's protocols andprocedures are followed for the annealing reaction.

SNP-IT Reaction

The extension reagent, which contains DNA polymerase, two terminatingnucleotides labeled with either fluorescein or biotin and two unlabeledterminators, is added to the SNP-IT well containing the annealedtemplate and primer complex. For SNP TSC0069085, ddCTP is labeled withfluorescein and ddTTP is labeled with biotin, and the unlabeledterminators are ddATP, and ddGTP. The manufacturer's protocols andprocedures are followed for the extension reaction.

The SNP specific base is incorporated by single base extension of theSNP-IT primer. Primers are washed manually or in a plate washer toremove unincorporated material. The manufacturer's protocols andprocedures are followed for the washing reaction.

Detection

Anti-fluorescein labeled with alkaline phosphatase (AP) is added to theplate and allowed to bind to any incorporated fluorescein labeledterminator. The manufacturer's protocols and procedures for the labelingreaction are followed.

The plates are washed, and then color development is performed usingpNPP as the detection substrate. The absorbance is read at 405 nm todetect yellow colored pNPP substrate followed by a washing step toremove pNPP detection reagents. The manufacturer's protocols andprocedures are followed for color development and washing steps.

Streptavidin labeled with horse radish peroxidase (HRP) is added to theplate and allowed to bind to any incorporated biotin labeled terminator.The manufacturer's protocols and procedures are followed for thelabeling reaction.

Following washing, color development is performed using TMB as thedetection substrate. The absorbance is read at 620 nm to detect bluecolored TMB substrate.

Analysis

Absorbance is plotted to generate a scatter plot from which genotypecalls are made. SNPs at which the maternal DNA is homozygous areidentified, and analyzed with the DNA isolated from the maternal plasma.

Analysis of DNA Isolated from Maternal Plasma

After the maternal DNA is analyzed and homozygous SNPs are identified,these SNPs are analyzed with the DNA isolated from the plasma. A lowcopy number of fetal genomes typically exists in the maternal plasma. Toincrease the copy number of the loci of interest, which are the SNPs atwhich the maternal DNA is homozygous, primers are designed to anneal atapproximately 130 bases upstream and 130 bases downstream of each lociof interest. This is done to reduce statistical sampling error that canoccur when working with a low number of genomes, which can influence theratio of one allele to another (see Example 11).

Design of Multiplex Primers

The primers are 12 bases in length. However, primers of any length canbe used including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36-45, 46-55, 56-65, 66-75, 76-85, 86-95, 96-105,106-115, 116-125, and greater than 125 bases. Primers are designed toanneal to both the sense strand and the antisense strand.

The maternal homozygous SNPs vary from sample to sample so definedsequences are not provided here. Primers are designed to anneal about130 bases upstream and downstream of the maternal homozygous SNPs. Theprimers are designed to terminate at the 3′ end in the dinucleotide “AA”to reduce the formation of primer-dimers. However, the primers can bedesigned to end in any of the four nucleotides and in any combination ofthe four nucleotides.

Multiplex PCR

Regions upstream and downstream of the maternal homozygous SNPs areamplified from the template genomic DNA using the polymerase chainreaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporatedherein by reference). This PCR reaction uses primers that annealapproximately 130 bases upstream and downstream of each loci ofinterest. The primers are mixed together and are used in a singlereaction to amplify the template DNA. This reaction is done to increasethe number of copies of the loci of interest, which eliminates errorgenerated from a low number of genomes.

For increased specificity, a “hot-start” PCR reaction is used. PCRreactions are performed using the HotStarTaq Master Mix Kit supplied byQIAGEN (catalog number 203443). The amount of template DNA and primerper reaction is optimized for each locus of interest. In this example,the 20 μl of plasma template DNA is used.

Two microliters of each forward and reverse primer, at concentrations of5 mM are pooled into a single microcentrifuge tube and mixed. Fourmicroliters of the primer mix is used in a total PCR reaction volume of50 μl (20 μl of template plasma DNA, 1 μl of sterile water, 4 μl ofprimer mix, and 25 μl of HotStar Taq. Twenty-five cycles of PCR areperformed. The following PCR conditions are used:

-   -   (1) 95° C. for 15 minutes;    -   (2) 95° C. for 30 second;    -   (3) 4° C. for 30 seconds;    -   (4) 37° C. for 30 seconds;    -   (5) Repeat steps 2-4 twenty-four (24) times;    -   (6) 72° C. for 10 minutes.

The temperatures and times for denaturing, annealing, and extension, areoptimized by trying various settings and using the parameters that yieldthe best results.

Other methods of genomic amplification can also be used to increase thecopy number of the loci of interest including but not limited to primerextension preamplification (PEP) (Zhang et al., PNAS, 89:5847-51, 1992),degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius, et al.,Genomics 13:718-25, 1992), strand displacement amplification using DNApolymerase from bacteriophage 29, which undergoes rolling circlereplication (Dean et al., Genomic Research 11:1095-99, 2001), multipledisplacement amplification (U.S. Pat. No. 6,124,120), REPLI-g™ WholeGenome Amplification kits, and Tagged PCR.

Purification of Fragment of Interest

The unused primers, and nucleotides are removed from the reaction byusing Qiagen MinElute PCR purification kits (Qiagen, Catalog Number28004). The reactions are performed following the manufacturer'sinstructions supplied with the columns. The DNA is eluted in 100 μl ofsterile water. 5 μl of each amplified loci is mixed together

SNP-IT Assay

The pooled DNA is assayed with the SNP-IT assay as described above. EachSNP is genotyped. SNPs located on chromosomes 13 and 21, wherein thematernal DNA is homozygous, and DNA isolated from the plasma isheterozygous are quantitated.

Quantification

The fluorescence intensity of each allele is quantitated. As discussedabove, the expected ratio of allele 1 to allele 2 is used to determinethe presence or absence of a chromosomal abnormality. If the maternalgenome is homozygous at SNP X (A/A), and the plasma DNA is heterozygousat SNP X (A/G), then the G represents the distinct fetal signal.

The intensity of the allele with the A nucleotide is quantitated and theintensity of the allele with the G nucleotide is quantitated. The ratioof G:A depends on the percentage of fetal DNA present in the maternalblood.

For example, if the sample contains 50% fetal DNA, then the expectedratio is 0.33 (1 fetal G allele/(2 maternal A alleles+1 fetal Aallele)). This ratio should be constant for all chromosomes that arepresent in two copies. The ratio that is obtained for SNPs on chromosome13 should be the same as the ratio that is obtained for chromosome 21.

However, if the fetal genome contains an additional copy of chromosome21, then the ratio for this chromosome will deviate from the expectedratio. The expected ratio for a Trisomy condition with 50% fetal DNA inthe maternal blood is 0.25. Thus, by analyzing SNPs wherein the maternalgenome is homozygous, and the DNA that is isolated from the plasma isheterozygous, fetal chromosomal abnormalities can be detected.

In this example, the terminator nucleotides are labeled with differentchemical moieties. However, using the methods described in thisapplication (see Example 6), the SNP-IT assay could be modified to allowdetection of both alleles with a single labeled terminator.

This example explained the use of Orchid's SNP-IT assay, but it notintended to limit the use of other techniques that rely on primerextension. Orchid's SNPstream 25K, as well accompanying softwareincluding but not limited to GetGenos™, QCreview™, and ValidGenos™, canalso be used to detect the presence of chromosomal abnormalities in thematernal blood. Additional information about these products can be foundat:

http://www.orchidbio.com/products/lsg/products/snpstream.asp.

Example 22

Fetal chromosomal abnormalities are determined by analyzing SNPs whereinthe maternal template DNA is homozygous and the template DNA obtainedfrom the plasma is heterozygous. Plasma that is isolated from blood of apregnant female contains both maternal template DNA and fetal templateDNA. Any number of SNP detection methods can be used to analyze thematernal and plasma DNA. In this example, SNPs are analyzed using theTaqMan® assay. However, other methods that rely on fluorogenic 5′nuclease assay can be used.

Collection of Blood Samples

In accordance with an IRB approved study, blood samples are collectedfrom pregnant women after informed consent is granted. Blood iscollected into 9 ml EDTA Vacuette tubes (catalog number NC9897284) and0.225 ml of 10% neutral buffered solution containing formaldehyde (4%w/v), is added to each tube, and each tube gently is inverted. The tubesare stored at 4° C. until ready for processing.

Any number of agents that impede cell lysis or stabilize cell membranescan be added to the tubes including but not limited to formaldehyde, andderivatives of formaldehyde, formalin, glutaraldehyde, and derivativesof glutaraldehyde, crosslinkers, primary amine reactive crosslinkers,sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfidereduction, carbohydrate reactive crosslinkers, carboxyl reactivecrosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP,APG, BASED, BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES,DFDNB, DMA, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS,HBVS, sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS or compounds listed in TableXXIII. Any concentration of agent that stabilizes cell membranes orimpedes cell lysis can be added. In a preferred embodiment, the agentthat stabilizes cell membranes or impedes cell lysis is added at aconcentration that does not impede or hinder subsequent reactions.

An agent that stabilizes cell membranes may be added to the maternalblood sample to reduce maternal cell lysis including but not limited toaldehydes, urea formaldehyde, phenol formaldehyde, DMAE(dimethylaminoethanol), cholesterol, cholesterol derivatives, highconcentrations of magnesium, vitamin E, and vitamin E derivatives,calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives,bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer Ahopane tetral phenylacetate, isomer B hopane tetral phenylacetate,citicoline, inositol, vitamin B, vitamin B complex, cholesterolhemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K,vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract,diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine,tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinccitrate, mexitil, dilantin, sodium hyaluronate, or polaxamer 188.

Isolation of Plasma and Maternal Cells

The blood is stored at 4° C. until processing. The tubes are spun at1000 rpm for ten minutes in a centrifuge with braking power set at zero.The tubes are spun a second time at 1000 rpm for ten minutes. Thesupernatant (the plasma) of each sample is transferred to a new tube andspun at 3000 rpm for ten minutes with the brake set at zero. Thesupernatant is transferred to a new tube and stored at −80° C.Approximately two milliliters of the “buffy coat,” which containsmaternal cells, is placed into a separate tube and stored at −80° C.

Isolation of DNA

DNA is isolated from the plasma sample using the Qiagen Midi Kit forpurification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA iseluted in 100 μl of distilled water. The Qiagen Midi Kit also is used toisolate DNA from the maternal cells contained in the “buffy coat.”

Identification of Homozygous Maternal SNPs TaqMan Assay

PE Biosystems has two instruments in its Sequence Detection Systemsproduct line, the ABI Prism® 7700 Sequence Detection System and theGeneAmp® 5700 Sequence Detection System. These real-time systemsallegedly are capable of detecting PCR products as they accumulateduring PCR and so enable the quantitation of DNA in the sample.

One chemistry available for use on the ABI PRISM® 7700 and GeneAmp® 5700detection systems is the fluorogenic 5′ nuclease assay, or the TaqMan®assay, which uses a fluorogenic probe to enable the detection of aspecific PCR product as it accumulates during PCR. PE Biosystems'patented fluorogenic probe design that incorporates the reporter due onthe 5′ end and the quencher on the 3′ end has assisted with the designof TaqMan probes.

The basis for PCR quantitation in the ABI 7700 instrument is tocontinuously measure PCR product accumulation using a dual-labeledfluorogenic oligonucleotide probe called a TaqMan® probe, which iscomposed of a short (20-25 bases) oligodeoxynucleotide that is labeledwith two different fluorescent dyes. On the 5′ terminus is a reporterdye and on the 3′ terminus is a quenching dye. This oligonucleotideprobe sequence is homologous to an internal sequence present in the PCRamplicon. When the probe is intact, energy transfer occurs between thetwo fluorophores and emission from the reporter is quenched by thequencher (Livak et al., PCR Methods and Applications, 4:357-362, 1995a;U.S. Pat. No. 5,538,848; U.S. Pat. No. 5,723,591).

During the extension phase of PCR, the probe is cleaved by 5′ nucleaseactivity of Taq polymerase thereby releasing the reporter from theoligonucleotide-quencher and producing an increase in reporter emissionintensity. The ABI Prism 7700 uses fiber optic systems which connect toeach well in a 96-well PCR tray format. The laser light source exciteseach well and a CCD camera measures the fluorescence spectrum andintensity from each well to generate real-time data during PCRamplification. The ABI 7700 Prism software examines the fluorescenceintensity of reporter and quencher dyes and calculates the increase innormalized reporter emission intensity over the course of theamplification. The results are then plotted versus time, represented bycycle number, to produce a continuous measure of PCR amplification. Toprovide precise quantification of initial target in each PCR reaction,the amplification plot is examined at a point during the early log phaseof product accumulation. This is accomplished by assigning afluorescence threshold above background and determining the time pointat which each sample's amplification plot reaches the threshold (definedas the threshold cycle number or CT). Differences in threshold cyclenumber are used to quantify the relative amount PCR target containedwithin each tube as described previously.

For SNP analysis, a TaqMan probe can be designed for each allele of theSNP. The reporter emission is used to determine the presence or absenceof each allele at the SNP. For example, for a SNP that can either beadenine or guanine, a TaqMan probe will be designed with a complementarynucleotide to the adenine and a separate TaqMan probe will be designedwith a complementary nucleotide to the guanine. The two TaqMan probescan be used in separate reaction vessels, which allows the amount of theadenine allele and the amount of the guanine allele to be calculated.

Primer and Probe Design

Primer and probes can be designed using the Primer Express® software.The probe is designed first, and then the primers are designed as closeas possible to the probe without overlapping it. Amplicons of 50-150base pairs are strongly recommended.

The primer and probes should be designed following the manufacturer'srecommendations. For both the primer and the probes, the G/C is in therange of 20-80%. The primer and probes are designed to avoid runs of anidentical nucleotide. This is especially true for guanine, where runs offour or more Gs should be avoided.

For the probe, the TM is about 68-70° C., and is designed so that thereis no guanine on the 5′ end. Also, the probe is designed so that thereare more C than G bases.

For the primers, the TM is about 58-60° C., and the primers are designedso that the five nucleotides at the 3′ end have no more than 2 G and/orC bases.

For example, representative primers and probes for SNP TSC0271628 (A/G),which is located on chromosome 21, are provided below:

Forward Primer (T_(M) of 60° C.) (SEQ ID NO: 657)5′ AGTCTTGTAATACGACAGTCTT 3′ Reverse Primer (T_(M) of 58° C.) (SEQ IDNO: 658) 5′ CCATATCAATCAGTACTCTTG 3′ TaqMan Probe A allele (T_(M) of68° C.; bold indicates variable nucleotide at SNP) (SEQ ID NO: 659)5′ CCTTTCCACTCATCCAAAGGTTG 3′ TaqMan Probe G allele (T_(M) of 70° C.;bold indicates variable nucleotide at SNP) (SEQ ID NO: 660)5′ CCTTTCCACTCATCCAGAGGTTG 3′

Information regarding the sequence surrounding SNPs is found at:http://www.snp.cshl.org. By independently varying forward and reverseprimer concentrations, the concentrations that provide optimal assayconditions can be identified. Primer concentration ranges of 50 nM-900nM are tested.

If the maternal DNA is homozygous for one allele, for example, adenine,then in the sample that contains the TaqMan probe specific for theguanine nucleotide, the reporter is not separated from the quencherbecause the TaqMan probe does not anneal to the template DNA. However,if the maternal DNA is homozygous, then the reporter will be separatedfrom the quencher in both samples containing the TaqMan probe specificfor the guanine allele and samples containing the TaqMan probe specificfor the adenine allele.

Reagent Solution

The polymerase recommended for the TaqMan Assay is AmpliTaq Gold DNApolymerase. It is thought that the use of AmpliTaq Gold DNA polymerasereduces the amount of non-specific product formation. The incorporationof AmpErase® Uracil n-glycosylase (UNG) and dUTP provide protectionagainst PCR carryover contamination. For PCR reactions, the TaqManUniversal PCR Master mix, which is a reagent designed to provide optimalperformance for TaqMan assays, is recommended by the manufacturer.

The TaqMan reaction buffer contains 5.5 mM MgCl2, 200 nM each of dATP,dCTP, dGTP, 400 nM dUTP, 0.5 U of uracyl DNA glycosylase, and 1.25 U ofAmpliTaq gold.

Thermal Cycling Parameters

PCR amplification and detection for all primer-probe combinations areperformed with the ABI 7700 Sequence Detection System. The recommendedcycling parameters for the TaqMan assay are provided below:

1) 50° C. for 2 min;

2) 95° C. for 10 min;

3) 95° C. for 15 sec;

4) 60° C. for 1 min;

5) Repeat steps 3-4 for 40 cycles.

TaqMan Quantitation

External standards are generated from know quantities of DNA containingan adenine nucleotide at SNP TSC0271628 and a guanine nucleotide at SNPTSC0271628, spanning 6 orders of magnitude (from 5×10⁰ to 5×10⁶ copies).The detection threshold is set at 10 times the standard deviation of themean baseline emission calculated for PCR cycles 3 to 15 (Shifts et al.,Applied and Environmental Microbiology, Vol. 67, No. 6, 2781-2789,2001). Standard curves relating the threshold cycle to DNAconcentrations are generated with the ABI Prism 7700 software (availablefrom Perkin Elmer).

SNPs at which the maternal DNA is homozygous are identified, and areanalyzed with the DNA isolated from the plasma sample.

Analysis of DNA Isolated from Maternal Plasma

After the maternal DNA is analyzed and homozygous SNPs are identified,these SNPs are analyzed with the DNA isolated from the plasma. A lowcopy number of fetal genomes typically exists in the maternal plasma. Toincrease the copy number of the loci of interest, which are the SNPs atwhich the maternal DNA is homozygous, primers are designed to anneal atapproximately 130 bases upstream and 130 bases downstream of each lociof interest. This is done to reduce statistical sampling error that canoccur when working with a low number of genomes, which can influence theratio of one allele to another (see Example 11).

Design of Multiplex Primers

The primers are 12 bases in length. However, primers of any length canbe used including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36-45, 46-55, 56-65, 66-75, 76-85, 86-95, 96-105,106-115, 116-125, and greater than 125 bases. Primers are designed toanneal to both the sense strand and the antisense strand.

The maternal homozygous SNPs vary from sample to sample so definedsequences are not provided here. Primers are designed to anneal about130 bases upstream and downstream of the maternal homozygous SNPs. Theprimers are designed to terminate at the 3′ end in the dinucleotide “AA”to reduce the formation of primer-dimers. However, the primers can bedesigned to end in any of the four nucleotides and in any combination ofthe four nucleotides.

Multiplex PCR

Regions upstream and downstream of the maternal homozygous SNPs areamplified from the template genomic DNA using the polymerase chainreaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporatedherein by reference). This PCR reaction uses primers that annealapproximately 130 bases upstream and downstream of each loci ofinterest. The primers are mixed together and are used in a singlereaction to amplify the template DNA. This reaction is done to increasethe number of copies of the loci of interest, which eliminates errorgenerated from a low number of genomes.

For increased specificity, a “hot-start” PCR reaction is used. PCRreactions are performed using the HotStarTaq Master Mix Kit supplied byQIAGEN (catalog number 203443). The amount of template DNA and primerper reaction is optimized for each locus of interest. In this example,the 20 μl of plasma template DNA is used.

Two microliters of each forward and reverse primer, at concentrations of5 mM are pooled into a single microcentrifuge tube and mixed. Fourmicroliters of the primer mix is used in a total PCR reaction volume of50 μl (20 μl of template plasma DNA, 1 μl of sterile water, 4 μl ofprimer mix, and 25 μl of HotStar Taq. Twenty-five cycles of PCR areperformed. The following PCR conditions are used:

-   -   (1) 95° C. for 15 minutes;    -   (2) 95° C. for 30 second;    -   (3) 4° C. for 30 seconds;    -   (4) 37° C. for 30 seconds;    -   (5) Repeat steps 2-4 twenty-four (24) times;    -   (6) 72° C. for 10 minutes.

The temperatures and times for denaturing, annealing, and extension, areoptimized by trying various settings and using the parameters that yieldthe best results.

Other methods of genomic amplification can also be used to increase thecopy number of the loci of interest including but not limited to primerextension preamplification (PEP) (Zhang et al., PNAS, 89:5847-51, 1992),degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius, et al.,Genomics 13:718-25, 1992), strand displacement amplification using DNApolymerase from bacteriophage 29, which undergoes rolling circlereplication (Dean et al., Genomic Research 11:1095-99, 2001), multipledisplacement amplification (U.S. Pat. No. 6,124,120), REPLI-g™ WholeGenome Amplification kits, and Tagged PCR.

It is important to ensure that the region amplified contains annealingsequences for the oligonucleotide probes in the BeadArray. Upon purchaseof the BeadArray service, each SNP and the primers used to analyze eachSNP are identified. With this knowledge, the multiplex primers aredesigned to encompass annealing regions for the primers in theBeadArray.

Purification of Fragment of Interest

The unused primers, and nucleotides are removed from the reaction byusing Qiagen MinElute PCR purification kits (Qiagen, Catalog Number28004). The reactions are performed following the manufacturer'sinstructions supplied with the columns. The DNA is eluted in 100 μl ofsterile water.

TaqMan Assay

The amplified DNA is assayed with the TaqMan assay as described above.Each SNP is genotyped. SNPs located on chromosomes 13 and 21, whereinthe maternal DNA is homozygous, and DNA isolated from the plasma isheterozygous are quantitated.

Quantification

The fluorescent intensity of the TaqMan allele specific probe isquantitated. As discussed above, the expected ratio of allele 1 toallele 2 is used to determine the presence or absence of a chromosomalabnormality. If the maternal genome is homozygous at SNP X (A/A), andthe plasma DNA is heterozygous at SNP X (A/G), then the G represents thedistinct fetal signal.

The fluorescent intensity of the allele with the A nucleotide isquantitated and the intensity of the allele with the G nucleotide isquantitated. The ratio of G:A depends on the percentage of fetal DNApresent in the maternal blood.

For example, if the sample contains 50% fetal DNA, then the expectedratio is 0.33 (1 fetal G allele/(2 maternal A alleles+1 fetal Aallele)). This ratio should be constant for all chromosomes that arepresent in two copies. The ratio that is obtained for SNPs on chromosome13 should be the same as the ratio that is obtained for chromosome 21.

However, if the fetal genome contains an additional copy of chromosome21, then the ratio for this chromosome will deviate from the expectedratio. The expected ratio for a Trisomy condition with 50% fetal DNA inthe maternal blood is 0.25. Thus, by analyzing SNPs wherein the maternalgenome is homozygous, and the DNA that is isolated from the plasma isheterozygous, fetal chromosomal abnormalities can be detected.

This example explained the use of the TaqMan assay, but it not intendedto limit the use of other techniques that employ 5′ nuclease activity.For example, the SYBR® Green I double stranded dye can also be used withthe ABI PRISM 7700 Sequence Detection System and the GeneAmp® 5700Sequence Detection system for determining the sequence of maternal andfetal DNA. The SYBR® Green I double stranded dye assay may be used todetect the presence of fetal chromosomal abnormalities in the maternalblood.

SYBR® Green I double stranded dye is a highly specific double-strandedDNA binding dye that allows the detection of product accumulation duringPCR. However, the SYBR® Green I double stranded dye assay detects alldouble stranded DNA including non-specific reaction products. Theadvantage of the SYBR® Green I double stranded dye assay is that it doesnot require a probe.

The same primers design parameters are recommended for both the TaqManAssay and the SYBR® Green I double stranded dye assay (see Primer Designsection in Example 21). The primer optimization parameters recommendedfor the TaqMan assay should also be followed for the SYBR® Green Idouble stranded dye assay. In addition, no template controls should alsobe run with the various concentrations of primers.

Furthermore, Applied Biosystems sell other products that may be used todetermine the sequence of maternal and fetal DNA including but notlimited to Assays-on-Demand™ SNP genotyping products, andAssays-by-Design^(SM) Service SNP genotyping products.

Having now fully described the invention, it will be understood by thoseof skill in the art that the invention can be performed with a wide andequivalent range of conditions, parameters, and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

All documents, e.g., scientific publications, patents and patentpublications recited herein are hereby incorporated by reference intheir entirety to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by referencein its entirety. Where the document cited only provides the first pageof the document, the entire document is intended, including theremaining pages of the document.

Example 23

Fetal chromosomal abnormalities are determined by analyzing SNPs whereinthe maternal template DNA is homozygous and the template DNA obtainedfrom the plasma is heterozygous. Plasma that is, isolated from blood ofa pregnant female contains both maternal template DNA and fetal templateDNA. Any number of SNP detection methods can be used to analyze thematernal and plasma DNA. In this example, SNPs are analyzed by ThirdWave Technologies' Invader™ Assay for Nucleic Acid Detection. However,other techniques that exploit and quantitate biological structuresformed in the presence of the correct sequence can be used.

Collection of Blood Samples

In accordance with an IRB approved study, blood samples are collectedfrom pregnant women after informed consent is granted. Blood iscollected into 9 ml EDTA Vacuette tubes (catalog number NC9897284) and0.225 ml of 10% neutral buffered solution containing formaldehyde (4%w/v), is added to each tube, and each tube gently is inverted. The tubesare stored at 4° C. until ready for processing.

Any number of agents that impede cell lysis or stabilize cell membranescan be added to the tubes including but not limited to formaldehyde, andderivatives of formaldehyde, formalin, glutaraldehyde, and derivativesof glutaraldehyde, crosslinkers, primary amine reactive crosslinkers,sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfidereduction, carbohydrate reactive crosslinkers, carboxyl reactivecrosslinkers, photoreactive crosslinkers, cleavable crosslinkers, AEDP,APG, BASED, BM(PEO)₃, BM(PEO)₄, BMB, BMDB, BMH, BMOE, BS3, BSOCOES,DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP, DSS, DST, DTBP, DTME, DTSSP, EGS,HBVS, sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS or compounds listed in TableXXIII. Any concentration of agent that stabilizes cell membranes orimpedes cell lysis can be added. In a preferred embodiment, the agentthat stabilizes cell membranes or impedes cell lysis is added at aconcentration that does not impede or hinder subsequent reactions.

An agent that stabilizes cell membranes may be added to the maternalblood samples to reduce maternal cell lysis including but not limited toaldehydes, urea formaldehyde, phenol formaldehyde, DMAE(dimethylaminoethanol), cholesterol, cholesterol derivatives, highconcentrations of magnesium, vitamin E, and vitamin E derivatives,calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives,bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer Ahopane tetral phenylacetate, isomer B hopane tetral phenylacetate,citicoline, inositol, vitamin B, vitamin B complex, cholesterolhemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K,vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract,diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine,tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinccitrate, mexitil, dilantin, sodium hyaluronate, or polaxamer 188.

Isolation of Plasma and Maternal Cells

The blood is stored at 4° C. until processing. The tubes are spun at1000 rpm for ten minutes in a centrifuge with braking power set at zero.The tubes are spun a second time at 1000 rpm for ten minutes. Thesupernatant (the plasma) of each sample is transferred to a new tube andspun at 3000 rpm for ten minutes with the brake set at zero. Thesupernatant is transferred to a new tube and stored at −80° C.Approximately two milliliters of the “buffy coat,” which containsmaternal cells, is placed into a separate tube and stored at −80° C.

Isolation of DNA

DNA is isolated from the plasma sample using the Qiagen Midi Kit forpurification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA iseluted in 100 μl of distilled water. The Qiagen Midi Kit also is used toisolate DNA from the maternal cells contained in the “buffy coat.”

Identification of Homozygous Maternal SNPs Third Wave TechnologiesInvader™ Assay

The Invader™ Assay, which was developed by Third Wave Technologies(Madison, Wis.), is an isothermal, “PCR-free” approach to the detectionand quantitative analysis of DNA. The Invader Assay produces andamplifies an unrelated signal only in the presence of the correct targetsequence. The Invader™ Assay relies on a thermostable member of thestructure-specific archeabacterial flap endonuclease (FEN) family, whichcleaves nucleic acid molecules at specific sites based on structurerather than sequence. When uses with structure forming probes for knownsequences, the enzymes cleave in a structure and targetsequence-specific manner. The nucleases used with Third WaveTechnologies' assays are referred to as “Cleavase®” enzymes.

The Invader™ Assay uses two target-specific oligonucleotides to createthe substrate complex recognized by Cleavase Enzymes (L. DeFrancesco,The Scientist, 12(21):16, 1998). The substrate complex is formed when anupstream Invader oligo and a downstream signal probe hybridize in tandemto the nucleic acid. The 3′ end of the Invader oligo must overlap thehybridization site of the signal probe by at least one base (Harringtonet at, Genes and Development, 8:1344-55, 1994). The 5′ end of the signalprobe has additional unpaired bases to form a 5′ flap. Cleavase enzymescleave the signal probe where it overlaps the Invader oligo, releasingthe 5′ arm. Reaction mixtures contain excess signal probe and arecarried out near the melting temperature of the probe. Many signalprobes can be cleaved for each copy of the target without temperaturecycling.

The overlap between the Invader oligo and the signal probe is important.A mismatch positioned at the site of the overlap will block the cleavageby disrupting the overlap, which may allow discrimination of SNPs andmutations.

The Invader assay utilizes two sequential cleavage steps. The 5′ arm ofthe signal probe released in the first reaction is not detecteddirectly. Rather, a secondary cleavage product is the actual source ofthe signal, detected by fluorescence resonance energy transfer (FRET).The primary cleavage product, which is the 5′ arm released in the firstreaction, is used as an Invader oligo that hybridizes to a supplied FRETprobe in the secondary reaction. The FRET probe is labeled with twodyes: a donor fluorophore and a quenching acceptor fluorophore. When thenuclease cleaves the secondary probe, the two fluorophores areseparated, quenching is eliminated, and the enhanced fluorescence signalfrom the donor dye is detected.

Probe Design

The following probes are designed to determine the sequence at SNPTSC1172576 (T/A), which is located on chromosome 13:

Invader Oligo for T allele: 5′ CATGCAGATATACCGCATAT 3′ (SEQ ID NO: 661)Invader Oligo for A allele: 5′ CATGCAGATATACCGCATAA 3′ (SEQ ID NO: 662)

Invader oligonucleotides are designed to be complementary to an 18-22base region immediately upstream of the signal probe, with an additionalone base at the 3′ end that “invades” the region hybridized to thesignal probe by one base.

Signal probe for T allele: GGTAGCATC TCTCAGCACAAGAG (SEQ ID NO: 663)Signal probe for A allele: GGTAGCATC ACTCAGCACAAGAG (SEQ ID NO: 664)

The signal probes are designed to contain a 3′ region that iscomplementary to the target sequence and a non-complementary 5′ arm (theunderlined sequence above) that is used for detection. The signal probesare labeled on the 5′ end with 6-carboxyfluorescein (TET),hexachloro-6-carboxyfluorescein (HEX), 6-carboxyfluorescein (FAM).However, the 5′ end can be labeled with any chemical moiety includingbut not limited to radioisotope, fluorescent reporter molecule,chemiluminescent reporter molecule, antibody, antibody fragment, hapten,biotin, derivative of biotin, photobiotin, iminobiotin, digoxigenin,avidin, enzyme, acridinium, sugar, enzyme, apoenzyme, homopolymericoligonucleotide, hormone, ferromagnetic moiety, paramagnetic moiety,diamagnetic moiety, phosphorescent moiety, luminescent moiety,electrochemiluminescent moiety, chromatic moiety, moiety having adetectable electron spin resonance, electrical capacitance, dielectricconstant or electrical conductivity, and combinations thereof

Both invader and signal probes are complementary to either the sense orthe antisense target DNA strand, depending on which results in theformation of the least number of predictable secondary structures.

PCR Amplification

A fragment of DNA that surrounds SNP TSC1172576 is amplified by PCR. Thesequence of the upstream and downstream primers is provided below:

Upstream Primer: 5′ TAGCAGAATCTCTCAT 3′ (SEQ ID NO: 665) DownstreamPrimer: 5′ AGAGTATCTCATTTGTT 3′ (SEQ ID NO: 666)

Amplification reactions are performed in a final volume of 100 μl ofcontaining 2 μl of genomic DNA, 35 pmol of each primer, 50 μm of eachdeoxynucleotide (Perkin-Elmer Applied Biosystems, Inc., Foster City,Calif.), 1×PCR buffer (20 mM Tris-Hcl, 50 mM KCl, 1.5 mM MgCl₂, 0.05%Tween-20, 0.05% NP40), 1 M betaine, 5% dimethylsulfoxide (DMSO), and 2.5U of Taq polymerase (Roche Boehringer Mannheim, Indianapolis, Ind.). PCRcycling conditions consist of an initial denaturation step at 95° C. for5 min, 30 cycles of denaturation at 95° C. for 1 min, annealing at 68°C. for 1 min, and extension at 72° C. for 1 min, and a final extensionat 72° C. for 5 min.

Invader Reaction

One microliter of each PCR product is added to 0.5 pmol of theappropriate Invader oligonucleotide, 10 ng human genomic DNA (PromegaCorp., Madison, Wis.) as the carrier, and mopholinepropanesulfonic acid(MOPS) buffer (pH 8.0) at a final concentration of 10 mM in a volume of7 μl. The mixtures are denatured for 5 min at 95° C. and then cooled toreaction temperature of 60° C. Invader reactions are initiated by theaddition of a mixture containing 30 ng of Cleavase VIII (Third WaveTechnologies, Inc., Madison, Wis.) 25 mM MgCl₂, and 10 pmol of theappropriate signal probe oligonucleotide in a volume of 3 μl. Reactionmixtures are incubated for 60 min. The reactions are terminated by theaddition of 10 μl of 95% formamide—10 mM EDTA (pH 8.0)—0.05% crystalviolet. Following termination, the reactions are diluted 1:10 inreagent-grade water. Samples of 2 μl are loaded and electrophoresed in a24% denaturing polyacrylamide gel (18 cm by 25.5 cm by mm) on anautomated fluorescence sequencing apparatus (model 377, PE-ABI). Thedata are collected using filter set C and processed with GeneScansoftware.

In addition, 5 μl of each sample is electrophoresed in 20% (acrylamideto bisacrylamide, 19:1) denaturing polyacrylamide gels at 20 W. Gelcassettes (20 cm by 20 cm by 0.5 mM) are scanned with a fluorescentscanner (FMBIO-100; Hitachi Corp, San Bruno, Calif.) by using a 585 nmfilter for TET and FLEX labeled probes and a 505 nm titter for FAMlabeled probes.

With the Invader assay, it is also possible to perform a second cleavagereaction where the released 5′ arm of the signal probe is hybridized toanother probe, and fluorescence resonance energy transfer (FRET) is usedto detect the presence of a specific nucleic acid. The manufacturer'sprotocols are followed when using the FRET probe.

The genotype at each SNP is determined by analyzing the fluorescenceintensity of each allele-specific signal probe. For example, for SNPTSC1172576, the presence of the T allele is determined by analyzing theamount of released 5′ signal probe using the signal probe from the Tallele signal probe (as described above). Likewise, the presence of theA allele is determined by analyzing the amount of released 5′ signalprobe from the A allele signal probe. The reactions can be performed ina single reaction vessel using two different chemical moieties, whichcan be analyzed under distinct conditions, or the A and T allelereactions can be performed in two different reaction vessels.

Analysis of DNA Isolated from Maternal Plasma

After the maternal DNA is analyzed and homozygous SNPs are identified,these SNPs are analyzed with the DNA isolated from the plasma. A lowcopy number of fetal genomes typically exist in the maternal plasma. Toincrease the copy number of the loci of interest, which are the SNPs atwhich the maternal DNA is homozygous, primers are designed to anneal atapproximately 130 bases upstream and 130 bases downstream of each lociof interest. This is done to reduce statistical sampling error that canoccur when working with a low number of genomes, which can influence theratio of one allele to another (see Example 11).

Design of Multiplex Primers

The primers are 12 bases in length. However, primers of any length canbe used including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36-45, 46-55, 56-65, 66-75, 76-85, 86-95, 96-105,106-115, 116-125, and greater than 125 bases. Primers are designed toanneal to both the sense strand and the antisense strand.

The maternal homozygous SNPs vary from sample to sample so definedsequences are not provided here. Primers are designed to anneal about130 bases upstream and downstream of the maternal homozygous SNPs. Theprimers are designed to terminate at the 3′ end in the dinucleotide “AA”to reduce the formation of primer-dimers. However, the primers can bedesigned to end in any of the four nucleotides and in any combination ofthe four nucleotides.

Multiplex PCR

Regions upstream and downstream of the maternal homozygous SNPs areamplified from the template genomic DNA using the polymerase chainreaction (PCR, U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporatedherein by reference). This PCR reaction uses primers that annealapproximately 130 bases upstream and downstream of each loci ofinterest. The primers are mixed together and are used in a singlereaction to amplify the template DNA. This reaction is done to increasethe number of copies of the loci of interest, which eliminates errorgenerated from a low number of genomes.

For increased specificity, a “hot-start” PCR reaction is used. PCRreactions are performed using the HotStarTaq Master Mix Kit supplied byQIAGEN (catalog number 203443). The amount of template DNA and primerper reaction is optimized for each locus of interest. In this example,the 20 μl of plasma template DNA is used.

Two microliters of each forward and reverse primer, at concentrations of5 mM are pooled into a single microcentrifuge tube and mixed. Fourmicroliters of the primer mix is used in a total PCR reaction volume of50 μl (20 μl of template plasma DNA, 1 μl of sterile water, 4 μl ofprimer mix, and 25 μl of HotStar Taq. Twenty-five cycles of PCR areperformed. The following PCR conditions are used:

-   -   (1) 95° C. for 15 minutes;    -   (2) 95° C. for 30 second;    -   (3) 4° C. for 30 seconds;    -   (4) 37° C. for 30 seconds;    -   (5) Repeat steps 2-4 twenty-four (24) times;    -   (6) 72° C. for 10 minutes.

The temperatures and times for denaturing, annealing, and extension, areoptimized by trying various settings and using the parameters that yieldthe best results.

Other methods of genomic amplification can also be used to increase thecopy number of the loci of interest including but not limited to primerextension preamplification (PEP) (Zhang et al., PNAS, 89:5847-51, 1992),degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius, et al.,Genomics 13:718-25, 1992), strand displacement amplification using DNApolymerase from bacteriophage 29, which undergoes rolling circlereplication (Dean et al., Genomic Research 11:1095-99, 2001), multipledisplacement amplification (U.S. Pat. No. 6,124,120), REPLI-g™ WholeGenome Amplification kits, and Tagged PER.

It is important to ensure that the region amplified contains annealingsequences for the oligonucleotide probes in the BeadArray. Upon purchaseof the BeadArray service, each SNP and the primers used to analyze eachSNP are identified. With this knowledge, the multiplex primers aredesigned to encompass annealing regions for the primers in theBeadArray.

Purification of Fragment of Interest

The unused primers, and nucleotides are removed from the reaction byusing Qiagen. MinElute PCR purification kits (Qiagen, Catalog Number28004). The reactions are performed following the manufacturer'sinstructions supplied with the columns. The DNA is eluted in 100 μl ofsterile water.

Invader Assay

The amplified DNA is assayed with the Invader assay as described above.Each SNP is genotyped. SNPs located on chromosomes 13 and 21, whereinthe maternal DNA is homozygous, and DNA isolated from the plasma isheterozygous are quantitated.

Quantification

The fluorescent intensity of the allele specific signal probe isquantitated. As discussed above, the expected ratio of allele 1 toallele 2 is used to determine the presence or absence of a chromosomalabnormality. If the maternal genome is homozygous at SNP X (A/A), andthe plasma DNA is heterozygous at SNP X (A/G), then the G represents thedistinct fetal signal.

The fluorescent intensity of the allele with the A nucleotide isquantitated and the intensity of the allele with the G nucleotide isquantitated. The ratio of G:A depends on the percentage of fetal DNApresent in the maternal blood.

For example, if the sample contains 50% fetal DNA, then the expectedratio is 0.33 (1 fetal G allele/(2 maternal A alleles+1 fetal Aallele)). This ratio should be constant for all chromosomes that arepresent in two copies. The ratio that is obtained for SNPs on chromosome13 should be the same as the ratio that is obtained for chromosome 21.

However, if the fetal genome contains an additional copy of chromosome21, then the ratio for this chromosome will deviate from the expectedratio. The expected ratio for a Trisomy condition with 50% fetal DNA inthe maternal blood is 0.25. Thus, by analyzing SNPs wherein the maternalgenome is homozygous, and the DNA that is isolated from the plasma isheterozygous, fetal chromosomal abnormalities can be detected.

1. A method for determining a sequence of a locus of interestcomprising: (a) amplifying a locus of interest on template DNA, whereinthe template DNA comprises a mixture of fetal DNA and maternal DNA andis obtained from a sample from a pregnant female, using a first andsecond primers, wherein the second primer contains a recognition sitefor a restriction enzyme such that digestion with the restriction enzymegenerates a 5′ overhang containing the locus of interest; (b) digestingthe amplified DNA with the restriction enzyme that recognizes therecognition site on the second primer; (c) incorporating a nucleotideinto the digested DNA of (b) by using the 5′ overhang containing thelocus of interest as a template; and (d) determining the sequence of thelocus of interest by determining the sequence of the DNA of (c).
 2. Themethod of claim 1, wherein said sample is selected from the groupconsisting of blood, serum, plasma, saliva, urine, tears, vaginalsecretion, sweat, lymph fluid, cerebrospinal fluid, mucosa secretion,peritoneal fluid, ascitic fluid, fecal matter, and body exudates.
 3. Themethod of claim 1, wherein said sample is blood.
 4. The method of claim3, wherein said blood is obtained from a human pregnant female when thefetus is at a gestational age selected from the group consisting of 0-4,4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, 40-44,44-48, 48-52, and more than 52 weeks.
 5. The method of claim 3, whereinsaid template DNA is obtained from plasma from said blood.
 6. The methodof claim 3, wherein said template DNA is obtained from serum from saidblood.
 7. The method of claim 1, wherein the restriction enzyme cuts DNAat a distance from the recognition site.
 8. The method of claim 1,wherein the recognition site is for a Type IIS restriction enzyme. 9.The method of claim 8, wherein the Type IIS restriction enzyme isselected from the group consisting of: Alw I, Alw26 I, Bbs I, Bbv I,BceA I, Bmr I, Bsa I, Bst71 I, BsmA I, BsmB I, BsmF I, BspM I, Ear I,Fau I, Fok I, Hga I, Pie I, Sap I, SSfaN I, and Sthi32 I.
 10. The methodof claim 1, wherein said method of amplification is PCR.
 11. The methodof claim 10, wherein an annealing temperature for cycle 1 of PCR isabout the melting temperature of the portion of the 3′ region of thesecond primer that anneals to the template DNA.
 12. The method of claim11, wherein an annealing temperature for cycle 2 of PCR is about themelting temperature of the portion of the 3′ region of the first primerthat anneals to the template DNA.
 13. The method of claim 12, wherein anannealing temperature for the remaining cycles of PCR is at about themelting temperature of the entire second primer.
 14. The method of claim1, wherein determining the sequence comprises a method selected from thegroup consisting of allele specific PCR, mass spectrometry,hybridization, primer extension, fluorescence resonance energy transfer(FRET), sequencing, Sanger dideoxy sequencing, DNA microarray, southernblot, slot blot, dot blot, and MALDI-TOF mass spectrometry.
 15. A methodfor determining a sequence of alleles of a locus of interest comprising:(a) amplifying alleles of a locus of interest on a template DNA, whereinthe template DNA comprises a mixture of fetal DNA and maternal DNA andis obtained from a sample from a pregnant female: using a first andsecond primers, wherein the second primer contains a recognition sitefor a restriction enzyme such that digestion with the restriction enzymegenerates a 5′overhang containing the locus of interest; (b) digestingthe amplified DNA with the restriction enzyme that recognizes therecognition site on the second primer; (c) incorporating nucleotidesinto the digested DNA of (b), wherein; (i) a nucleotide that terminateselongation, and is complementary to the locus of interest of an allele,is incorporated into the 5′ overhang of said allele, and (ii) anucleotide complementary to the locus of interest of a different alleleis incorporated into the 5′ overhang of said different allele, and saidterminating nucleotide, which is complementary to a nucleotide in the 5′overhang of said different allele, is incorporated into the 5′ overhangof said different allele; (d) determining the sequence of the alleles ofa locus of interest by determining the sequence of the DNA of (c). 16.The method of claim 15, wherein said sample is selected from the groupconsisting of blood, serum, plasma, saliva, urine, tears, vaginalsecretion, sweat, lymph fluid, cerebrospinal fluid, mucosa secretion,peritoneal fluid, ascitic fluid, fecal matter, and body exudates. 17.The method of claim 15, wherein said sample is blood.
 18. The method ofclaim 17, wherein said template DNA is obtained from plasma from saidblood.
 19. The method of claim 17, wherein said template DNA is obtainedfrom serum from said blood.
 20. The method of claim 15, wherein therecognition site is for a Type IIS restriction enzyme.
 21. The method ofclaim 20, wherein the Type IIS restriction enzyme is selected from thegroup consisting of: Alw I, Alw26 I, Bbs I, Bbv I, BceA I, Bmr I, Bsa I,Bst71 I, BsmA I, BsmB I, BsmF I, BspM I, Ear I, Fau I, Fok I, Hga I, PieI, Sap I, SSfaN I, and Sthi32 I.